BIOLOGY  LIBRARY 


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3563  LIFE  SCIENCES  BUILDING 


r.  LEGGE,  M.  D. 

6  ROBLE  ROAD 
BERKELEY,   CALIF. 


WORKS  BY 
CHARLES    F.    BOLDUAN,    M.D. 

PUBLISHED  BY 

JOHN  WILEY  &  SONS 


Immune  Sera. 

A  concise  exposition  of  the  main  facts  and 
theories  of  infection  and  immunity,  Fourth 
edition,  rewritten.  By  Charles  F.  Bolduan,  M.D. 
12mo,  ix  +  226  pages.  Cloth,  $1.50. 

TRANSLATIONS. 
The  Suppression  of  Tuberculosis. 

Together  with  Observations  concerning  Phthisio- 
genesis  in  Man  and  Animals,  and  Suggestions  con- 
cerning the  Hygiene  of  Cow  Stables  and  the  Pro- 
duction of  Milk  for  Infant  Feeding,  with  Special 
Reference  to  Tuberculosis.  By  Professor  E.  von 
Behring,  University  of  Marburg.  Authorized 
Translation  by  Charles  F.  Bolduan,  M.D.  12mo, 
vi  +  85  pages.  Cloth,  $1.00. 

Manual  of  Serum  Diagnosis. 

By  Doctor  O.  Rostoski,  University  of  Wurzburg. 
Authorized  Translation  by  Charles  F.  Bolduan, 
M.D.  12mo,  vi  +  86  pages.  Cloth,  $1.00. 

Studies  in  Immunity. 

By  Professor  Paul  Ehrlich.  Translated  by  Charles 
F.  Bolduan,  M.D.  Second  Edition,  Revised  and 
Enlarged.  8vo,  xi  + 712  pages.  Cloth,  $6.00. 


IMMUNE  SERA 

A   CONCISE   EXPOSITION    OF    THE 
MAIN    FACTS    AND   THEORIES   OF 

INFECTION  AND   IMMUNITY 


BY 

DR.  CHARLES  FREDERICK  BOLDUAN 

BACTERIOLOGIST,    RESEARCH    LABORATORY,  DEPARTMENT   OF    HEALTH 
CITY  OF  NEW  YORK 


FOURTH  EDITION,  REWRITTEN  AND  ENLARGED 
FIRST    THOUSAND 


NEW   YORK: 

JOHN  WILEY  &  SONS 

LONDON:    CHAPMAN  &  HALL,  LIMITED 

1911 


9tl 


67 


COPYRIGHT,  1907,  1908,  1911. 
BT  CHARLES  FREDERICK   BOLDUAN 


THE  SCIENTIFIC   PRESS 

ROBERT   DRUMMOND   AND    COMPANY 

BROOKLYN,    N.   Y. 


TO 
HERMANN  M.   BIGGS,  M.D.,  LL.D. 

PIONEER    IN    SPECIFIC    SERUM    THERAPY    AND    SERUM 

DIAGNOSIS    IN    AMERICA,      FOUNDER     OF    THE 

FIRST  MUNICIPAL  SERUM  LABORATORY 

IN    THE    WORLD,      THIS    VOLUME 

IS  DEDICATED  AS  A  TOKEN 

OF  GREAT  ADMIRATION 

AND  ESTEEM 


PREFACE  TO  THE   FOURTH   EDITION 


THE  favorable  reception  accorded  to  the  previous 
editions  of  this  book,  together  with  the  fact  that 
our  knowledge  of  the  subject  has  increased  con- 
siderably in  the  past  few  years,  has  led  the  author 
to  undertake  a  thorough  revision  of  the  work. 

In  its  first  edition,  in  1904,  this  book  dealt  only 
with  certain  antibodies  whose  discovery  had  aroused 
a  great  deal  of  scientific  interest,  namely,  hsemoly- 
sins,  cytotoxins,  and  precipitins.  To  this  was 
added,  in  subsequent  editions,  a  discussion  of  anti- 
toxins, agglutinins,  and  opsonins.  All  these  topics 
were  naturally  embraced  under  the  title  "  Immune 
Sera."  In  the  present  (fourth)  edition,  while  the 
old  title  has  been  retained,  the  scope  of  the  sub- 
ject matter  has  been  greatly  extended,  so  that  now 
there  is  presented  an  exposition  of  the  main  facts 
of  infection  and  immunity. 

It  is  but  natural  that  any  discussion  of  the 
immunity  reactions  should  center  about  the  ingen- 
ious side-chain  theory  of  Ehrlich,  which  has  domi- 
nated the  work  in  this  field.  Its  heuristic  value 


vi  PREFACE 

has  unquestionably  been  very  great.  At  the  same 
time  it  cannot  be  doubted  that  some  of  the  deduc- 
tions from  the  theory  have  led,  here  and  there, 
to  strained  conceptions  which  apparently  violate 
established  biological  facts.  While  presenting 
Ehrlich's  views  at  length,  therefore,  the  author 
has  endeavored  to  bring  out  clearly  just  why  and 
wherein  certain  other  investigators  differ.  The  aim 
of  the  book  has  been  to  present  a  broad,  clear  outline 
of  the  main  facts  and  theories  concerning  infection 
and  immunity,  and  while  this  may  perhaps  have 
led  to  the  omission  of  some  really  excellent  studies, 
it  was  felt  best  not  to  confuse  the  beginner  with  a 
mass  of  apparently  contradictory  observations. 

CHARLES  BOLDUAN. 

NEW  YORK,  April,  1911. 


CONTENTS 


PAGE 

Antitoxins i 

HISTORICAL ; i 

PRESENT    METHOD    OF    PRODUCING    DIPHTHERIA    ANTI- 
TOXIN    2 

PRODUCTION  OF  DIPHTHERIA  TOXIN 2 

IMMUNIZING  THE  ANIMALS 3 

COLLECTING  THE  SERUM 4 

TESTING  THE  STRENGTH  OF  THE  SERUM 5 

EHRLICH'S  THEORY  FOR  PRODUCTION 6 

TOXINS,  TOXOIDS 6 

RECEPTORS 9 

WEIGERT'S  OVERPRODUCTION  THEORY 10 

EXPERIMENTAL  EVIDENCE  FOR  EHRLICH'S  THEORY...  .  13 

ANTIGENS  OR  HAPTINS 16 

NATURE  OF  ANTITOXINS  IN  GENERAL 17 

TOXINS  AND   OTHER  POISONOUS  CELL  DERIVATIVES  IN 

GENERAL 20 

RELATIONS  BETWEEN  TOXIN  AND  ANTITOXIN 22 

"L0"and   "Lt" 23 

PARTIAL  SATURATION   METHOD   OF  STUDYING  TOXINS 

— TOXONS,  TOXOIDS 23 

EHRLICH'S  "  POISON  SPECTRA  " 24 

VIEWS  OF  ARRHENIUS,  BORDET,  AND  OTHERS 28 

Agglutinins — 32 

THE  PHENOMENON 32 

H^MAGGLUTININS 34 

ISOAGGLUTININS 35 

PURPOSE  OF  AGGLUTINATION 36 

HISTORICAL 37 

PFAUNDLER'S  REACTION  (THREAD  REACTION).  . 38 

vii 


CONTENTS 


PAGE 

NATURE  OF  AGGLUTININS  AND  OF   THE  AGGLUTINATION 

REACTION  ......................................  39 

AGGLUTINOIDS  ......................................  39 

GROUP  AGGLUTININS  :  ...............................  43 

ABSORPTION  METHODS  FOR  DIFFERENTIATING  BETWEEN 

A  MIXED  AND  A  SINGLE  INFECTION  ................  46 

FORMATION   OF  AGGLUTININS   ACCORDING  TO   THE   ^IDE- 
CHAIN  THEORY,  RECEPTORS  OF  FIRST,  SECOND,  AND 

THIRD  ORDER  ..................................  47 

Bacteriolysins  and  Haemolysins  ..................  5o 

HISTORICAL  ........................................  50 

FFEIFFER'S  PHENOMENON  ............................  51 

HAEMOLYSIS  ........................................  52 

NATURE  OF  H^MOLYTIC  SERA  ....................  .  .  54 

ANALOGY    BETWEEN    THE    BACTERIOLYTIC    AND    HVEMO- 

LYTIC  PROCESSES  ...............................  57 

EHRLICH  AND  MORGENROTH  ON  THE  NATURE  OF  HAEMO- 

LYSIS ..........................................  58 

THEIR  THREE  CLASSIC  EXPERIMENTS  ................  59 

NOMENCLATURE  ...................................  63 

ROLE  OF  THE  IMMUNE  BODY  .............  .-  .........  64 

ON  WHAT  THE  SPECIFICITY  DEPENDS  ................  65 

DIFFERENCE  BETWEEN  A  SPECIFIC  SERUM  AND  A  NOR- 

MAL ONE  .........................  ..  ............  66 

DIVERGING  VIEWS  OF  EHRLICH  AND  BORDET  ...........  67 

THE  SIDE-CHAIN  THEORY  APPLIED  TO  THESE  BODIES.  .  .  68 

MULTIPLICITY  OF  COMPLEMENTS  ......................  70 

THE      BORDET-GENGOU      PHENOMENON;        NEISSER- 

SACHS  BLOOD  TEST  .............................  71 

NORMAL  SERUM,    ITS   H^MOLYTIC   AND   BACTERIOLYTIC 

,     ACTION  ........................................  73 

ACTIVE  AND  INACTIVE  NORMAL  SERUM  .......  '.  ......  75 

ACTION  NOT  ENTIRELY  SPECIFIC  ....................  77 

MULTIPLICITY  OF  THE  ACTIVE  SUBSTANCES  ...........  78 

DIFFERENCE  BETWEEN  A  NORMAL  AND  A  SPECIFIC  IM- 

MUNE SERUM  ...................................  79 

NATURE     OF     THE     IMMUNE     BODY  —  PARTIAL     IMMUNE 

BODIES  OF  EHRLICH.  .  82 


CONTENTS  {x 

PAGE 

METCHNIKOFF'S  VIEWS 84 

SUPPORT  FOR  EHRLICH'S  VIEW 85 

ANTIH^MOLYSINS:     THEIR    NATURE — ANTI-COMPLEMENT 

OR  ANTI-IMMUNE  BODY 86 

ANTI-COMPLEMENT 88 

FLUCTUATIONS   IN   THE  AMOUNT   OF   THE  ACTIVE   SUB- 
STANCES IN  SERUM 91 

SOURCE     OF     THE     COMPLEMENTS — LEUCOCYTES     AS     A 

SOURCE — OTHER  SOURCES 93 

STRUCTURE  OF  THE  COMPLEMENTS — COMPLEMENTOIDS.  .  .  94 

ISOLYSINS AUTOLYSINS ANTI-ISOLYSINS 97 

DEFLECTION  OF  COMPLEMENT 100 

PRACTICAL  VALUE  OF  ANTI-BACTERIAL  SERA 104 

Precipitins 107 

DEFINITIONS 107 

BACTERIAL  PRECIPITINS 108 

LACTOSERUM — OTHER  SPECIFIC  PRECIPITINS 108 

SPECIFICITY  OF  THE  PRECIPITINS 109 

NATURE  OF  THE  PRECIPITINS in 

PRACTICAL  APPLICATION 112 

THE  WASSERMANN-UHLENHUTH  BLOOD  TEST 113 

IMMUNIZING  THE  ANIMALS 114 

COLLECTING  THE  SERUM 115 

THE  TEST 116 

APPEARANCE  OF  THE  REACTION 117 

DELICACY  OF  THE  PRECIPITIN  TEST 118 

OTHER  APPLICATIONS  OF  THE  PRECIPITIN  TEST ,  .  .  1 18 

ANTIPRECIPITINS — ISOPRECIPITINS 119 

Cy  totoxins 121 

DEFINITION,  LEUCOTOXIN — NATURE  OF  THE  CYTOTOXIN 

ANTICYTOTOXIN 121 

NtvUROTOXIN 122 

SPERMOTOXIN 123 

COMMON  RECEPTORS 124 

CYTOTOXIN  FOR  EPITHELIUM 124 

CYTOTOXINS  BY  THE  USE  OF  NUCLEOPROTEIDS 125 

Opsonins 127 

HISTORICAL 127 


X  CONTENTS 

PAGE 

BACTERIOTROPIC  SUBSTANCES 129 

OPSONINS  DISTINCT  ANTIBODIES. 130 

STRUCTURE  OF  OPSONINS 130 

THE  OPSONIC  INDEX 131 

TECHNIQUE 131 

VALUE  OF  THE  OPSONIC  MEASUREMENTS 133 

vSnake  Venoms  and  their  Antisera 137 

THE  VENOMS 137 

ANTIVENIN'S 139 

Anaphylaxis 141 

HISTORICAL 141 

THE  PHENOMENON 142 

SERUM  RASHES 143 

THEORIES  OF  ANAPHYLAXIS 144 

ALLERGY 147 

SUPPOSED  RELATION  TO  PRECIPITIN  ACTION 148 

PATHOLOGY  OF  ANAPHYLACTIC  SHOCK 150 

RELATION  OF  ANAPHYLAXIS  TO  SERUM  THERAPY 151 

Infection  and  Immunity 154 

INFECTION 154 

THE  INFECTING  AGENT 154 

ANAPHYLATOXIN 157 

RESISTANCE  AGAINST   INFECTION 158 

NATURAL  IMMUNITY 159 

ACQUIRED  IMMUNITY 160 

MECHANISM  OF  IMMUNITY 162 

RELATION  OF  ANAPHYLAXIS  TO  IMMUNITY 163 

IMMUNITY  REACTION  OF  THE  PART  OF  BACTERIA 165 

ATREPSY 166 

Bacterial  Vaccines 169 

HISTORICAL.  .  . 169 

METHODS  OF  ACTIVE  IMMUNIZATION 171 

TREATMENT  WITH  VACCINES 173 

THE  VACCINES 174 

DOSES :....' 175 

RESULTS 176 


CONTENTS  xi 

PAGE 

Leucocyte  Extracts  in  the  Treatment  of  Infec- 
tions   177 

THEORY 177 

PREPARATION  OF  THE  EXTRACTS 177 

APPLICATION  AND  RESULTS 173 

Principles  Underlying  Treatment  of  Syphilis  with 

Salvarsan 179 

PARASITOTROPISM  AND  ORGANOTROPISM 179 

CHEMORECEPTORS 181 

Appendices 185 

A.  THE  WASSERMANN  TEST  FOR  SYPHILIS 185 

B.  NOGUCHI'S  MODIFIED  WASSERMANN  REACTION 200 

C.  BLOOD     EXAMINATION     PREPARATORY     TO     TRANS- 
FUSION   204 

D.  THE  CONGLUTINATION  REACTION 207 

THE  MUCH-HOLZMANN  COBRA  VENOM  REACTION  .  ..  208 

THE  MEIOSTAGMIN  REACTION 208 

WEIL'S  COBRA  VENOM  TEST  IN  SYPHILIS 210 

ANTITRYPSIN  DETERMINATIONS.  .'..                              .  212 


IMMUNE  SERA 


ANTITOXINS 

Historical.  — The  researches  of  Buchner1  in  1889 
had  shown  that  the  serum  of  animals  artificially 
immunized  against  a  certain  bacterium  possessed 
marked  bactericidal  properties  for  that  particular 
organism.  In  studying  immunity  on  animals  which 
had  been  successfully  immunized  against  diphtheria 
infection,  Behring,2  working  in  Koch's  laboratory 
was  struck  by  the  fact  that  in  these  animals 
living  virulent  diphtheria  bacilli  were  often  demon- 
strable in  the  scab  at  the  site  of.  injection  several 
weeks  after  the  infection,  and  furthermore  that  the 
blood  serum  of  the  animals  did  not  possess  bacteri- 
cidal properties.  In  a  study  published  in  1890 
Behring  showed  that  the  serum  of  rabbits  arti- 
ficially immunized  against  diphtheria  was  able  to 
confer  a  specific  immunity  against  diphtheria  infec- 
tions in  other  animals.  He  also  demonstrated  that 
such  a  serum  could  be  used  therapeutically  to  cure 
an  infection  already  in  progress.  Such  a  serum 

1  Buchner,  Centralblatt  Bacteriologie,  Vol.  v,  1889.     Archiv. 
f.  Hygiene,  Vol.  x.  1890. 

2  Behring    &    Kitasato,    Deutsche   med.    Wochenschrift,  No. 
49,     1890. 

I 


2  IMMUNE  SERA 

was  not  bactericidal,  and  retained  its  therapeutic 
power  for  a  considerable  time.  He  believed  that 
the  action  of  the  serum  was  effected  by  a  neu- 
tralization of  the  bacterial  toxin  by  an  "  antitoxic 
serum  constituent."  The  action  was  strictly  specific, 
an  antitoxic  serum  obtained  after  a  diphtheria 
infection  protected  only  against  diphtheria;  one 
derived  from  a  tetanus  animal,  only  against  tetanus. 
Subsequently  Behring  and  Knorr  showed  that  im- 
munization could  be  effected  with  bacterial-free 
filtrates  of  tetanus  cultures  and  that  the  serum  thus 
produced  protected  not  only  against  tetanus 
infection  but  against  poisoning  by  the  toxic  prod- 
ucts of  the  bacilli.  After  considerable  experi- 
mental work  Behring  and  his  collaborators  devised 
an  effective  method  of  immunizing  Lheep  and 
certain  other  animals  against  diphtheria  and  against 
tetanus  and  so  produced  antitoxic  sera  in  con- 
siderable amounts. 

The  following  account  taken  from  Park  shows  the 
present  methods  of  producing  diphtheria  antitoxin. 

Production  of  the  Diphtheria  Toxin.  — A  strong 
diphtheria  toxin  should  be  obtained  by  taking  a  very 
virulent  culture  and  growing  it  in  broth  which  is  about 
8  cc.  normal  soda  solution  per  liter  above  the  neutral 
point  to  litmus.  The  culture  fluid  should  be  in  com- 
paratively thin  layers  and  in  large-necked  Erlenmeyei 
flasks,  so  as  to  allow  of  a  free  access  of  air;  the  tem- 
perature should  be  about  35°  to  36°  C.  The  culture, 
after  a  weeks  growth,  is  removed  from  the  incubator, 


ANTITOXINS  3 

and  having  been  tested  for  purity  by  microscopic  and 
culture  tests  is  rendered  sterile  by  the  addition  of  10 
per  cent  of  a  5  per  cent  solution  of  carbolic  acid.  After 
48  hours  the  dead  bacilli  have  settled  on  the  bottom  of 
the  jar  and  the  clear  fluid  is  filtered  through  ordinary 
sterile  filter  paper  and  stored  in  full  bottles  in  a 
cold  place  until  needed.  Its  strength  is  then  tested  by 
giving  a  series  of  guinea  pigs  carefully  measured 
amounts.  Less  than  o.oi  cc.  when  injected  hypoder- 
mically  should  kill  a  250  gram  guinea  pig. 

Immunizing  the  Animals.  — The  horses  used  should 
be  young,  vigorous,  of  fair  size,  and  absolutely  healthy. 
Vicious  habits,  such  as  kicking,  etc.,  make  no  difference, 
except,  of  course,  to  those  who  handle  the  animals. 
The  horses  are  severally  injected  with  an  amount  of 
toxin  sufficient  to  kill  five  thousand  guinea  pigs  of  250 
grams  weight  (about  20  cc.  of  strong  toxin).  After 
from  three  to  five  days,  so  soon  as  the  fever  reaction 
has  subsided,  a  second  subcutaneous  injection  of  a 
slightly  larger  dose'  is  given.  With  the  first  three 
injections  of  toxin  10,000  units  of  antitoxin  are  given. 
If  antitoxin  is  not  mixed  with  the  first  doses  of  toxin 
only  one-tenth  of  the  doses  advised  is  to  be  given. 
At  intervals  of  from  five  to  eight  days  increasing  injec- 
tions of  pure  toxin  are  made  until  at  the  end  of  two 
months  from  ten  to  twenty  times  the  original  amount 
is  given.  There  is  absolutely  no  way  of  judging  which 
horses  will  produce  the  highest  grades  of  antitoxin. 
Very  roughly  those  horses  which  are  extremely  sensi- 
tive, and  those  which  react  hardly  at  all  are  the  poorest, 
but  even  here  there  are  exceptions.  The  only  way, 
therefore,  is  at  the  end  of  six  weeks  or  two  months  to 
bleed  the  horses  and  test  their  serum.  If  only  high 
grade  serum  is  wanted  all  the  horses  that  give  less 


4  IMMUNE  SERA 

than  150  units  per  cc.  are  discarded.  If  moderate 
grades  only  are  desired,  all  that  yield  100  units  may  be 
retained.  The  retained  horses  receive  steadily  in- 
creasing doses,  the  rapidity  of  the  increase  and  the 
interval  of  time  between  the  doses  (three  days  to  one 
week)  depending  somewhat  on  the  reaction  following 
the  injection,  an  elevation  of  temperature  of  more  than 
3°  F.  being  undesirable.  At  the  end  of  three  months 
the  antitoxic  serum  of  all  the  horses  should  contain 
over  300  units  and  in  about  10  per  cent  as  much  as  800 
units  per'cc.  Very  few  horses  ever  give  over  1000 
units,  and  none  so  far  has  given  as  much  as  2000  units 
per  cc.  The  very  best  horses,  if  pushed  to  their 
limit  continue  to  furnish  blood  of  gradually  decreasing 
strength.  If  every  nine  months  an  interval  of  three 
months'  freedom  from  inoculations  is  given,  the  best 
horses  furnish  high  grade  serum  during  their  periods  of 
treatment  for  from  two  to  four  years. 

Collecting  the  Serum.  — In  order  to  obtain  the  serum 
the  blood  is  withdrawn  from  the  jugular  vein  by  means 
of  a  sharp-pointed  canula  which  is  plunged  through  the 
vein  wall,  a  slit  having  been  made  in  the  skin.  The 
blood  is  carried  by  a  sterile  rubber  tube  attached  to  the 
canula,  into  large  Erlenmeyer  flasks  and  allowed  to 
clot,  the  flasks,  however  being  placed  in  a  slanting 
position  before  clotting  has  commenced.  The  serum 
is  drawn  off  after  4  days  by  means  of  sterile  glass  and 
rubber  tubing,  and  is  stored  in  large  flasks  in  a  refrige- 
rator. From  this  as  needed  small  vials  are  filled. 
The  vials  and  their  stoppers,  as  indeed  all  the  utensils 
used  for  holding  the  serum,  must  be  absolutely  sterile 
and  every  possible  precaution  must  be  taken  to  avoid 
contamination  of  the  serum.  An  antiseptic  may  be 
added  as  a  preservative,  but  is  not  necessary.  Diph- 


ANTITOXINS  5 

theria  antitoxin,  when  stored  in  vials  and  kept  in  a  cool 
place  away  from  light  and  air  contains  within  10  per 
cent  of  its  original  strength  for  at  least  two  months; 
after  that  it  can  be  used  by  allowing  for  a  maximum 
deterioration  of  3  per  cent  for  each  month. 

Testing  the  Strength  of  the  Antitoxin.  — This  is  carried 
out  as  follows:  Six  guinea  pigs  .are  injected  with  mix- 
tures of  toxin  and  antitoxin.  In  each  of  the  mixtures 
there  is  100  times  the  amount  of  a  toxin  (similar  to 
that  adopted  as  the  standard)  which  will  kill  a  250 
grams  on  an  average  in  96  hours.  In  each  of  the 
mixtures  the  amount  of  antitoxin  varies;  for  instance, 
No.  i  would  contain  0.002  cc.  serum;  No.  2,  0.003  cc. ; 
No.  3,  0.004  cc. ;  No.  4,  0.005  cc->  etc-  ^  at  tne  end  °f 
the  fourth  day  Nos.  i,  2  and  3  were  dead  and  Nos.  4, 
5  and  6  were  alive  we  would  consider  the  serum  to 
contain  200  units  of  antitoxin  for  each  cubic  centi- 
meter. When  we  mix  only  ten  fatal  doses  of  toxin 
with  one-tenth  of  the  amount  of  antitoxin  used  with 
100  fatal  doses,  the  guinea  pig  must  remain  well.  The 
mixed  toxin  and  antitoxin  must  remain  together  for 
fifteen  minutes  before  injecting. 

Behring's  publication  was  followed  in  the  next 
two  years  by  considerable  work  along  these  lines, 
valuable  contributions  being  made  by  Aronson,1 
Roux,  and  Martin,2  Wernicke,3  Knorr 4  and  -others. 
The  statements  of  Behring  as  to  the  strict  specifi- 
city of  the  antitoxins  were  fully  confirmed.  Certain 

1  Berliner  med.   Gesellschaft,  Sitzung,   Dec.  21,   1892.     Also 
Berliner  Klin.  Wochenschrift,  1893  and  1894. 
3  Roux  and  Martin,  Annal.  Pasteur    1894. 
8  Behring  and  Wernicke,  Zeitsch.   Hygiene,  1892.     Vol.  xi. 
Behring  and  Knorr,  Zeitsch.  f.  Hygiene,  1893.     Vol.  xii. 


6  IMMUNE  SERA 

observations  by  Buchner 1  and  by  Roux  and  Martin 
threw  doubt,  however,  on  the  correctness  of  Beh- 
rings  view  that  the  toxin  was  neutralized  by  the 
specific  serum  just  as  a  base  was  neutralized  by  an 
acid.  It  was  claimed,  for  example,  that  the  specific 
serum  acted  mainly  on  the  body  cells  causing  them 
to  become  non-susceptible  to  the  poison  in  question. 
Various  theories  were  formulated  to  account  for  the 
production  of  the  antitoxins,  their  specificity,  etc., 
but  of  them  all  only  one  has  at  all  maintained  itself. 
This,  is  the  so-called  side-chain  theory,  which  was 
formulated  by  Ehrlich2  in  1897. 

Ehrlich's  Side-Chain  Theory.  —  Originally  the 
side-chain  theory  was  applied  by  Ehrlich  only  to 
the  production  of  the  specific  antitoxins,  i.e.,  sub- 
stances in  the  blood,  which  act  not  only  on  the 
living  bacteria,  but  also  and  especially  on  their 
dissolved  toxins.  Later  on  he  extended  it  so  as 
to  apply  also  to  the  formation  of  specific  bacteri- 
cidal and  haemolytic  substances  in  the  serum  of 
animals  treated  with  living  bacteria  or  with  animal 
cells. 

Toxins  —  Toxoids  —  Special  Function  of  the  Side 
Chains.  —  The  basis  of  the  theory  is  the  fact  that 
poison  and  counter-poison,  toxin  and  antitoxin, 
combine  directly  in  any  given  quantity.  This 
combination  always  occurs  in  definite  proportions 

1  Buchner,  Miinchener  med.  Wochenschrift,  1894. 
1  Ehrlich,  Klinisches  Jahrbuch,  1897. 


ANTITOXINS  7 

following  the  laws  of  chemical  combination;  and, 
still  following  those  laws,  is  slower  at  lower  tem- 
peratures than  at  higher,  stronger  in  concentrate  J. 
than  in  dilute  form.  Ehrlich  could  further  show 
that  each  poison  for  which  by  the  process  of  immun- 
izing one  can  develop  a  counter-poison  possesses 
two  groups  which  are  concerned  in  the  combina- 
tion with  the  counter-poison  or  antitoxin.  One  of 
these,  the  so-called  haptophore  group,  is  the  combin- 
ing group  proper;  the  other,  the  toxophore  group, 
is  the  carrier  of  the  poison.  A  poison  molecule, 
therefore,  might  lose  the  one,  the  toxophore,  and 
still  be  capable  by  means  of  its  haptophore  group 
of  combining  with  antitoxin.  Such  a  modified 
poison,  which  because  of  the  loss  of  the  toxophore 
group  can  hardly  be  called  a  poison,  but  which  still 
possesses  the  power  to  combine  with  antitoxin, 
Ehrlich  calls  a  toxoid.  Toxoids  may  be  produced 
spontaneously  in  old  poisons  through  decomposi- 
tion of  the  poison  molecule,  or  they  may  be  pro- 
duced artificially  by  causing  certain  destructive 
agents  such  as  heat  or  chemicals  to  act  on  bacterial 
poisons.  The  toxophore  group  is  a  very  delicate 
one  and  much  more  readily  decomposed  than  the 
combining  (haptophore)  group.  Ehrlich  reasoned 
that  in  order  for  a  poison  to  be  toxic  to  an  organ- 
ism, i.e.,  in  order  that  the  toxophore  group  be  able 
to  act  destructively  on  a  cell,  it  is  necessary  for  the 
haptophore  group  of  the  poison  to  combine  with 


8  IMMUNE  SERA 

the  cell.  IS  In  every  living  cell,"  Ehrlich  says, 
"  there  must  exist  a  dominating  body  [Leistungs 
Kern]  and  a  number  of  other  chemical  groups  or 
side  chains.  These  groups  have  the  greatest  variety 
of  function,  but  especially  those  of  nutrition  and 
assimilation." 

The  side  chains,  then,  according  to  this  author, 


—  toxophore  group 


,   POISON   MOLECULE 


FlG.  I 

are  able  to  combine  with  the  greatest  variety  of 
foreign  substances  and  convert  these  into  nourish- 
ment suitable  to  the  requirements  of  the  active 
central  body.  They  are  comparable  to  the  pseudo- 
podia  of  the  lower  animals,  which  engulf  food  par- 
ticles and  assimilate  the  same  for  the  immediate 
use  of  the  organism.  In  order  that  any  substance 


ANTITOXINS  9 

may  combine  with  these  side  chains  it  is  necessary 
that  certain  very  definite  relations  exist  between 
the  combining  group  of  the  substance  and  that 
of  the  side  chain.  Using  the  well-known  simile  of 
Emil  Fischer,  the  relation  must  be  like  that  of  lock 
and  key,  i.e.,  the  two  groups  must  fit  accurately. 
Hence  not  every  substance  will  fit  all  the  side 
chains  of  an  organism.  It  will  combine  only  with 
those  for  which  it  possesses  a  fitting  group. 

Receptors  —  Weigert's  Overproduction  Theory.  — 
This  doctrine  of  the  chemistry  of  the  organism's 
metabolism  Ehrlich  applied  to  the  action  of  toxins 
and  antitoxins.  '  The  toxin,"  he  said,  "  can  act 
only  when  its  haptophore  group  happens  to  fit  to 
one  of  the  side  chains,"  or  receptors,  as  he  now  pre- 
fers to  call  them.  As  a  result  of  this  combination, 
the  toxophore  group  is  able  to  act  on  the  cell  and 
injure  it.  If  we  take  as  an  example  tetanus,  in 
which  all  the  symptoms  are  due  to  the  central  ner- 
vous system,  the  side-chain  theory  assumes  that 
the  haptophore  group  of  the  tetanus  poison  fits 
exactly  and  is  combined  with  the  side  chain  or 
receptors  of  the  central  nervous  system.  Other 
experiments,  which  we  will  not  reproduce  here, 
have  shown  us  unquestionably  that  the  action  of 
the  antitoxins  depends  on  the  fact  that  this  com- 
bines with  the  haptophore  group  of  the  poison  and 
so  satisfies  the  latter 's  affinity.  Ehrlich,  therefore, 
concluded  that  the  antitoxin  is  nothing  else  than 


10  IMMUNE  SERA 

the  side  chains  or  receptors  which  are  given  off  by 
the  cells  and  thrust  into  the  circulation.  The  way 
in  which  these  side  chains  or  receptors  are  thrust 
off  as  a  result  of  the  immunizing  process,  Ehrlich 
explains  by  means  of  Weigert1  s  Overproduction 
Theory. 

At  the  meeting  of  German  Naturalists  and 
Physicians  held  at  Frankfurt  in  1896,  Weigert  *  in 
discussing  regeneration,  advanced  an  hypothesis  the 
essential  features  of  which  are  that  physiological 
structure  and  function  depend  upon  the  equilibrium 
of  the  tissues  maintained  by  virtue  of  mutual 
restraint  between  their  component  cells ;  that  destruc- 
tion of  a  single  integer  or  group  of  integers  of  a 
tissue  or  a  cell  removes  a  corresponding  amount  of 
restraint  at  the  point  injured,  and  therefore  destroys 
equilibrium  and  permits  of  the  abnormal  exhibi- 
tion of  bioplastic  energies  on  the  part  of  the  remain- 
ing uninjured  components,  which  activity  may  be 
viewed  as  a  compensating  hyperplasia;  that  hyper- 
plasia  is  not,  therefore,  the  direct  result  of  external 
irritation,  and  cannot  be,  since  the  action  of  the 
irritant  is  destructive  and  is  confined  to  the  cells 
or  integers  of  cells  that  it  destroys,  but  occurs 
rather  indirectly  as  a  function  of  the  surrounding 
uninjured  tissues  that  have  been  excited  to  bio- 
plastic  activity  through  the  removal  of  the  restraint 

1  Weigert,  Verhandlungen  der  Ges.  deutscher  Naturforscher 
iind  Aerzte,  1896 


ANTITOXINS  II 

hitherto  exerted  by  the  cells  destroyed  by  the 
irritant;  and,  finally,  when  such  bioplastic  activity 
is  called  into  play  there  is  always  hypercompen- 
sation  —  i.e.  there  is  more  plastic  material  gene- 
rated than  is  necessary  to  compensate  for  the 
loss. 

Ehrlich  points  out  that  owing  to  the  combination 
of  the  toxin  with  the  side  chain  of  a  cell,  these 
side  chains  are  practically  lost  to  the  cell ;  that  the 
latter  or  its  fellows  now  produces  new  side  chains  to 
replace  this  loss,  but  that  this  production  always 
goes  so  far  as  to  make  a  surplus  of  side  chains ;  that 
these  side  chains  are  thrown  off  by  the  cell  as 
unnecessary  ballast,  and  then  circulate  in  the  blood 
as  antitoxin.  The  same  substances,  therefore,  which 
when  part  of  the  cell  combine  with  the  haptophore 
group  of  the  toxin,  enabling  that  to  act  on  the  cell, 
when  circulating  free  in  the  blood  combine  with 
and  satisfy  this  haptophore  group  of  the  toxin, 
and  prevent  the  poison  from  combining  with  and 
damaging  the  cells  of  the  organism. 

It  does  not  follow  from  Ehrlich's  theory  that  the 
antitoxin  is  produced  by  the  same  set  of  cells  whose 
injury  by  the  toxin  gives  rise  to  the  particular 
clinical  symptoms.  Thus  we  might  believe  that 
although  in  tetanus  the  cells  of  the  central  nervous 
s}^stem  give  rise  to  the  characteristic  symptoms,  cells 
entirely  apart  from  these,  e.g.,  in  the  bone  marrow, 
might  be  the  main  source  of  the  antitoxin.  The 


12  IMMUNE  SERA 

fact  that  we  appreciate  symptoms  from  only  one 
organ  is,  obviously,  no  proof  that  other  tissues 
have  been  unaffected. 

It  may  be  well  here  to  call  attention  to  another 
rather  common  misconception  regarding  the  pro- 
duction of  antitoxin,  namely  that  the  body  cells 
have  to  become  educated,  so  to  speak,  to  produce 
the  antitoxin.  This,  it  is  believed,  is  effected  by 
giving  gradually  increasing  doses  of  toxin.  As  a 
matter  of  fact  the  reason  for  this  gradual  increase 
in  the  dose  injected  is  quite  different.  The  object 
in  view  is  the  administration  of  an  enormously 
large  dose  of  toxin,  one  that  will  engage  the  recep- 
tors of  many  cells,  The  previous  injections  have 
brought  about  some  production  of  antitoxin  and 
this  partially  neutralizes  some  of  the  toxin  in- 
jected, making  it  possible  to  give  a  larger  dose  than 
before.  If  one  gives  at  the  outset  a  large  amount  of 
toxin,  partially  neutralized  by  antitoxin,  one  will 
produce  an  amount  of  antitoxin  equal  to  that 
ordinarily  obtained  in  response  to  the  same  quan- 
tity of  unaltered  toxin  given  as  the  tenth  or 
twentieth  injection  of  a  series.  Park  and  Atkinson 
for  example,  injected  a  fresh  horse  with  one  litre 
of  a  toxin  neutralized  ij  times  for  guinea  pigs. 
At  the  end  of  a  week  the  horse  had  produced  a  serum 
containing  60  units  per  cc.  When  the  toxin  was 
neutralized  6  fold  no  antitoxin  whatever  was  pro- 
duced. 


ANTITOXINS  13 

Experimental  Evidence  for  Ehrlictis  Theory.  — 
According  to  Ehrlich,  then,  the  formation  of  specific 
antibodies  must  proceed  in  three  stages: 

1.  The  binding  of  the  haptophore  group  to  the 
receptor. 

2.  The    increased    production    of    the    receptors 
following  this  binding. 

3.  The  thrusting-off  of  these  increased  receptors 
into  the  blood. 

So  far  as  the  first  point  is  concerned  Wassermann  * 
showed  that  with  tetanus,  in  which,  as  is  well 
known,  all  the  symptoms  are  referable  to  the  cen- 
tral nervous  system,  tetanus  toxin  was  bound  by 
central  nervous  system  substance  in  vitro.  A 
mixture  of  tetanus  poison  and  normal  central 
nervous  system  was  innocuous  to  animals,  showing 
that  certain  substances  present  in  the  central 
nervous  system  combine  with  and  thus  satisfy  the 
affinity  of  the  haptophore  group  of  the  poison. 
This  of  course  prevents  the  latter  from  combining 
with  any  cells  of  the  organism.  Organs  other  than 
the  central  nervous  system  do  not  possess  this 
property  of  combining  with  tetanus  poison,  just 
as  the  central  nervous  system  is,  on  the  contrary, 
incapable  of  combining  with  diphtheria  poison, 
which  clinically  does  not  show  any  pronounced 
affinity  for  the  central  nervous  system. 

Wassermann  2  also  believes  recently  to  have  given 

1  Wassermann  and  Takaki,   Berliner  Klin.  Wochenschr,  1898, 
1  Wassermann,  New  York  Medical  Journal,  1904. 


14  IMMUNE  SERA 

experimental  proof  of  the  second  and  third  points, 
the  increased  production  of  the  receptors  and  their 
thrusting  off.  For  this  purpose  he  employed  a 
tetanus  poison  which  he  had  kept  for  about  eight 
years,  and  which  was  originally  very  poisonous. 
In  the  course  of  years,  however,  owing  to  the 
damaging  action  of  light,  of  oxidation,  etc.,  it  had 
become  so  weak  that  it  was  no  longer  toxic  at  all. 
Injections  of  one  cc.  into  a  guinea  pig  produced 
no  tetanus.  Nevertheless  the  haptophore  group 
remained  intact,  as  could  readily  be  proved,  for 
this  non-poisonous  tetanus  toxin  was  still  able  to 
bind  tetanus  antitoxin,  i.e.  thrust-off  receptors.  On 
injecting  rabbits  with  this  nOn-poisonous  tetanus 
toxoid  in  increasing  doses,  and  then  examining  the 
blood  serum  of  the  animal  he  found  not  a  trace  of 
tetanus  antitoxin.  This  absence  could  have  either 
of  two  causes:  It  might  be  that  the  toxoid  no 
longer  produced  any  physiological  effect  whatever 
in  the  organism;  or  although  it  still  caused  an 
increase  in  the  receptors,  these  increased  receptors 
remained  in  the  organs  (sessile)  and  were  not 
thrust  off  into  the  blood.  In  order  to  decide  this 
question  Wassermann  first  determined  the  exact 
quantity  of  fresh  tetanus  toxin  which  constituted  a 
fatal  dose  for  guinea  pigs.  He  reasoned  that  if 
he  injected  first  the  toxoid,  and  shortly  after,  say 
in  one  or  two  hours,  the  fresh  toxin,  he  should  in 
such  an  animal  have  to  increase  the  fatal  dose, 


ANTITOXINS  1 5 

i.e.  more  tetanus  toxin  should  be  required  to  kill 
this  animal  than  a  normal  one,  because  owing  to 
the  previous  toxoid  injection  part  of  the  cells  sus- 
ceptible to  tetanus  toxin  would  already  have  been 
occupied.  Provided  Ehrlich's  theory  were  correct, 
so  that  this  binding  of  the  toxoid  really  occurred, 
the  conditions  should  be  entirely  different  when, 
instead  of  injecting  the  toxin  shortly  after  the 
toxoid,  he  waited  somewhat  longer,  one  to  three 
days,  and  then  injected  the  fresh  tetanus  toxin 
In  that  case  Weigert's  law  should  come  into  play 
and  the  receptors  have  commenced  to  increase 
in  number,  i.e.  the  organ  should  now  possess  more 
sensitive  groups  than  before.  This  would  manifest 
itself  in  such  fashion  that  in  contrast  to  the  first 
experiment  the  fatal  dose  of  fresh  tetanus  toxin 
could  now  be  decreased ;  in  other  words  a  small  dose 
would  now  tetanize  the  animal  in  a  shorter  time. 

As  a  matter  of  fact  Wassermann's  experiments 
yielded  exactly  the  results  deduced  theoretically. 
He  injected  a  guinea  pig  with  some  of  the  non-- 
poisonous toxoid  and  then,  an  hour  later,  with 
tetanus  toxin.  He  found  that  much  more  toxin 
was  required  to  kill  this  animal  than  a  normal 
guinea  pig  of  equal  size.  When,  on  the  contrary, 
he  waited  one  to  three  days,  it  was  found  that  then 
a  dose  of  tetanus  toxin  which  would  not  even 
tetanize  a  normal  guinea  pig  was  sufficient  to  kill 
this  one. 


16  IMMUNE  SERA 

It  will  be  seen  that  in  the  above  experiments 
the  completely  non-poisonous  toxoid,  although  it 
effected  an  increased  production  of  receptors,  did 
not  cause  their  thrusting-off.  The  serum  of  the 
rabbit  treated  with  toxoid  contained  no  antitoxin 
whatever.  Wassermann  concludes  from  this  and 
other  experiments  that  the  thrusting-off  cannot  be 
a  function  of  the  haptophore  group,  and  that 
something  additional  is  required.  This  "  some- 
thing," he  claims  is  a  function  of  the  toxophore 
group.  It  may  be  stated  that  Von  Dungern  has 
also  published  experiments  (with  majaplasm)  point- 
ing to  the  existence  of  the  second  stage,  the  stage  of 
sessile  receptors. 

Antigens  or  Haptins.  — •  It  has  been  found  that 
it  is  impossible  to  produce  any  immunity  against 
all  poisons,  e.g.  strychnine  or  morphine.  Accord- 
ing to  Ehrlich  these  simpler  chemical  molecules  do 
not  enter  into  a  true  chemical  combination  with 
the  tissues,  but  form  rather  a  kind  of  solid  solution, 
a  loose  combination  with  the  cells,  so  that  they  can 
again  be  abstracted  from  these  cells  by  all  kinds  of 
solvents,  e.g.  by  shaking  out  with  ether  or  chloro- 
form. The  point  can  perhaps  be  likened  to  the 
difference  between  saccharin  and  sugar.  Both  sub- 
stances taste  sweet,  but  despite  this  similarity  in 
their  physiological  action  they  behave  very  dif- 
ferently toward  the  cells  of  the  organism.  Sac- 
charin simply  passes  through  the  organism  without 


ANTITOXINS  17 

entering  into  a  firm  combination,  i.e.  without  being 
assimilated,  and  is  therefore  no  food.  Its  sweeten- 
ing action  is  a  mere  contact  effect  on  the  cells 
sensitive  to  taste.  Sugar,  on  the  contrary,  is 
actually  bound  by  the  cells,  assimilated  and  burnt, 
and  so  is  a  true  food.  Until  recently  it  was  believed 
that  the  simpler  chemical  substances  could  not 
excite  the  production  of  antibodies.  Ford  and 
Abel 1  have  however  been  able  to  show  that  toad 
stool  poison,  a  true  toxin,  against  which  an  anti- 
toxin can  be  produced  is  chemically  a  glucoside. 

As  we  shall  subsequently  see  it  is  possible  to 
immunize  the  animal  body  against  a  large  number 
of  substances,  including  not  only  such  cell  products 
as  ferments,  toxins  and  venoms,  but  also  cells  of 
the  greatest  variety,  bacteria,  dissolved  proteids,  etc. 
All  these  substances,  therefore,  must  possess  hapto- 
phore  groups  able  to  combine  with  the  side  chains 
or  receptors  in  the  animal  body.  Collectively, 
we  speak  of  such  substances  as  antigens  or  haptins. 

Nature  of  Antitoxins  in  General.  —  But  little  is 
known  concerning  the  constitution  of  antitoxins, 
for  we  do  not  know  them  apart  from  serum  or 
serum  constituents.  It  seems  probable  that  they 
are  proteid  in  character,  but  this  has  not  been 
positively  decided.  It  has  been  found  that  like 
the  globulins  they  are  quite  resistant  to  the  action 
of  trypsin,  but  are  acted  on  by  pepsin-hydrochloric 

1  Ford  and  Abel,  Journal  of  Biological  Chemistry,  Vol.  ii,  1907 


1 8  IMMUNE  SERA 

acid.  In  general  they  withstand  a  fair  degree  of 
heat,  certainly  far  more  than  the  toxins.  Anti- 
toxins are  to  be  regarded  as  inactive  substances, 
effecting  merely  a  blocking  of  the  haptophore 
group  of  the  corresponding  toxin.  They  do  not 
act  on  the  toxins  destructively.  This  is  indicated 
by  experiments  of  Wassermann  on  pyocyaneus  toxin, 
and  of  Calmette  and  Morgenroth  1  on  snake  venom, 
which  showed  that  in  the  toxin-antitoxin  com- 
bination, the  toxin  could  again  manifest  itself  after 
the  antitoxin  had  been  destroyed.  The  antitoxins 
therefore  are  not  ferment-like  substances.  As  far 
back  as  1897  attempts  were  made  to  determine  the 
chemical  nature  of  the  antitoxins.  In  that  year 
Belfanti  and  Carbone 2  found  that  the  antitoxin 
was  precipitated  with  the  globulins  of  the  serum  by 
means  of  magnesium  sulphate.  Dieudonne 3  had 
previously  shown  that  the  proteids  thrown  out  of 
solution  by  acetic  and  carbonic  acids  contained 
none  of  the  antitoxin.  In  1901  Atkinson4  showed 
that  the  globulins  increase  markedly  in  the  serum 
of  horses  as  the  antitoxic  strength  increases.  The 
most  recent  work  on  this  subject  is  that  of  Gibson,5 
who  shows  that  if  the  ammonium  sulphate  precipi- 

1  Morgenroth,  Berlin,  klin.  Wochenschr.  1905. 

2  Beifanti   and   Carbone,    Centralblatt    Bacteriologie    (Ref.), 
Vol.  xxiii,  1898. 

3  Dieudonne,  Arbeiten  a.d.  kaiserl.     Gesundheitsamte.     VoL 
xiii,  1897. 

4  Atkinson,  Jour.  Exper.  Medicine,  Vol.  i,  1901. 

5  Gibson,  Journ.  Biological  Chemistry,  Vol.  i,  1906. 


ANTITOXINS  1 9 

tate  (globulins,  nucleo-proteids,  etc.)  is  treated  with 
saturated  sodium  chloride  solution,  practically  all 
the  antitoxic  fraction  passes  into  solution.  Gibson's 
was  the  first  really  practicable  method  of  concentrat- 
ing the  antitoxin.  By  means  of  it  solutions  of 
antitoxic  globulin  could  easily  be  made  to  contain 
1500  units  per  cc.  Continuing  Gibson's  work, 
Banzhaf  discovered  that  if  the  antitoxic  serum  or 
plasma  was  heated  to  57°  for  18  hours,  there  was 
a  change  of  a  considerable  portion  of  the  soluble 
globulins  (soluble  in  Nad  solution)  into  insoluble 
globulins.  The  antitoxin  remained  unchanged. 
This  procedure,  therefore,  permits  of  a  still  greater 
elimination  of  the  non-antitoxic  proteids. 

Gibson  has  recently  studied  the  possibility  of 
differentiating  other  antibodies  by  means  of  their 
precipitation  characteristics.  He  believes  that  a 
differentiation  of  the  antibodies  into  those  precip- 
itated with  the  pseudo  globulins  and  with  the 
euglobulin  fractions,  according  to  the  Hofmeister 
classification,  is  based  on  a  misconception  of  the 
application  of  ammonium  sulphate  in  separating 
proteids  by  their  precipitation  characters.  While 
there  seem  to  be  some  differences  in  the  dis- 
tribution of  the  antibodies  in  individual  specific 
sera  in  comparative  experiments,  this  is  not  so 
absolute  as  maintained  by  Pick l  and  others.  Gib- 
son's work  on  the  fractionating  of  poly  agglutina- 

1  Pick,  Beitiage  z.  chem.  PhysioL  u-  PathoL,  VoL  i,  1901. 


20  IMMUNE  SERA 

tive  serum  shows  that  no  separation  of  the  several 
antibodies  developed  in  an  individual  serum  is 
possible.  In  the  case  of  antitoxic  sera  both  Gibson 
and  Ledingham  find  that  in  goat  serum  the  antitoxin 
is  not  invariably  associated  with  the  euglobulin 
fraction  as  maintained  by  Pick,  but  shows  the  same 
solubilities  as  that  in  horse  serum. 

Toxins  and  other  Poisonous  Cell  Derivatives,  in 
General.  — •  Soon  after  bacteriology  had  demon- 
strated the  etiological  connection  between  bacteria 
and  disease,  the  conviction  gained  ground  that  it 
was  less  the  actual  destruction  wrought  by  the 
bacteria  directly,  than  the  injury  produced  by  their 
chemical  products  that  gave  rise  to  the  lesions  in 
the  infectious  diseases.  Brieger,  especially,  was 
one  of  the  first  to  direct  attention  to  the  probable 
existence  of  specific  poisons  in  the  bacteria.  He 
isolated  a  number  of  well  defined  chemical  sub- 
stances called  ptomaines,  most  of  which  were  highly 
toxic.  Subsequent  study,  however,  showed  that 
these  were  not  the  specific  bacterial  poisons.  The 
latter,  the  true  toxins  are  something  quite  different 
as  we  shall  see  in  a  moment.  Still  later  other 
substances  were  isolated  from  bacteria,  and  these 
were  termed  toxalbumins.  We  now  know  that 
some  of  these  were  identical  with  the  true  toxins, 
but  that  others  were  entirely  unrelated. 

What  then  are  the  true  toxins?  A  number  of 
pathogenic  bacteria,  when  grown  in  pure  culture, 


ANTITOXINS  21 

produce  dissolved  poisons  in  the  culture  fluid. 
These  poisons  are  neither  ptomaines  nor  proteid 
substances;  their  chemical  nature  is  still  absolutely 
unknown.  They  are  extremely  sensitive  to  exter- 
nal influences,  especially  against  heat,  and  in  many 
ways  are  very  analogous  to  ferments.  Physio- 
logically the  toxins  are  extremely  poisonous,  far 
beyond  that  of  any  of  the  ordinary'  well  known 
poisons,  and  this  poisonous  action  manifests  itself 
only  after  a  certain  latent  period  known  as  the 
period  of  incubation.  Finally  one  of  the  funda- 
mental properties  of  the  toxins  is  their  ability 
to  excite,  in  the  organism  attacked,  antitoxins 
directed  specifically  against  them,  so  that  for  every 
true  toxin  there  is  a  corresponding  antitoxin. 

In  addition  to  these  bacterial  toxins  we  know 
of  other  poisonous  substances  possessing  similar 
characteristics.  Among  these  are  the  "  zootoxins," 
-  snake  venoms,  spider  and  toad  poisons,  the 
toxin  of  eel  blood,  and  the  "  phytotoxins," 
ricin,  crotin,  abrin,  etc.  It  may  be  mentioned  that 
some  of  these  are  of  somewhat  more  complex  con- 
stitution than  the  ordinary  bacterial  toxins.  Ricin, 
for  example,  appears  to  possess  one  haptophore 
group  but  two  ergophore  groups,  a  toxic  and  an 
agglutinating  one.  In  the  case  of  the  snake 
venoms  it  is  not  yet  definitely  known  whether 
they  are  haptins  of  the  first  order  or  of  the 
second.  (See  page  49.) 


22  IMMUNE   SERA 

The  Relations  Existing  between  Toxin  and  Anti- 
toxin. —  The  exact  nature  of  the  toxin-antitoxin 
reaction  has  long  been  the  subject  of  study  and  has 
given  rise  to  considerable  discussion.  For  obvious 
reasons  most  of  the  work  has  been  done  with 
diphtheria  and  tetanus  toxins  and  their  antitoxins. 
In  order  to  give  the  reader  some  conception  of 
the  diverging  views  of  various  authorities  we  shall 
devote  a  few  pages  to  a  brief  study  of  ths  diphtheria 
toxin-antitoxin  reaction. 

During  the  earlier  years  of  toxin-antitoxin  in- 
vestigations the  filtered  or  sterilized  bouillon,  in 
which  the  diphtheria  bacillus  had  grown  and  pro- 
duced its  "  toxin,"  was  supposed  to  require  for 
its  neutralization  an  amount  of  antitoxin  directly 
proportional  to  its  toxicity  as  tested  in  guinea  pigs. 
Thus,  if  from  one  bouillon  culture  ten  fatal  doses 
of  "  toxin"  were  required  to  neutralize  a  certain 
quantity  of  antitoxin,  it  was  believed  that  ten 
fatal  doses  from  every  culture,  without  regard  to 
ths  way  in  which  it  had  been  produced  or  preserved, 
would  also  neutralize  the  same  amount  of  antitoxin. 
Upon  this  belief  was  founded  the  Behring-Ehrlich 
definition  of  an  antitoxin  unit.1 

The  results  of  tests  by  different  experimenters 
of  the  same  antitoxic  serum,  but  with  different  diph- 

1  This  unit  was  "  ten  times  the  amount  of  antitoxic  serum 
necessary  to  just  protect  a  250  gramme  guinea  pig  against 
ten  fatal  doses  of  the  toxin  " 


ANTITOXINS 


theria  toxins,  proved  this  opinion  to  be  incorrect. 
Ehrlich  1  deserves  the  credit  for  first  clearly  per- 
ceiving and  calling  attention  to  this  fact.  He 
obtained  from  various  sources  twelve  toxins  and 
compared  their  neutralizing  value  upon  antitoxin; 
these  tests  gave  interesting  and  important  in- 
formation. The  following  table  gives  the  results 
in  four  of  his  toxins  and  well  illustrates  the  point  in 
question : 


Smallest  num- 

Fatal doses  re- 

ber of  fatal 
doses  of  toxic 

quired  to 
"  completely 

Estimated 

bouillon  re- 

neutralize "  one 

L+  minus 

Serial 

minimal  fatal 

quired  to  kill  a 

antitoxin  unit 

Num- 
ber. 

dose  for 
250  gm. 
guinea  pigs. 

2sogm.  guinea 
pig  within 
5  days  when 
mixed  with  one 

as  determined 
by   the  health 
of  the  guinea 
pig  remaining 

L(>  in 
fatal 
doses. 

Remarks. 

antitoxin  unit. 

unaffected. 

("Lt  Ehrlich.") 

("  LO  Ehrlich.") 

A 

o  .  009  cc. 

39-4 

33-4 

6 

Old;        deterio- 

rated from  0.003 

;o  o.  009. 

B 

0.0165  cc 

76.3 

54-4 

22 

Fresh         toxin, 

preserved    with 

:ricresol. 

C 

o.  039  cc. 

123. 

108. 

15 

A     numbei      of 

"resh      cultures, 

grown  at  37°  C. 

four    and    eight 

days. 

D 

0.0025  cc. 

100 

5° 

5° 

Tested  immedi- 

ately   after    its 

x 

withdrawal. 

It  was  natural  to  suppose,  as  the  early  investi- 
gators did,  that  a  just  neutral  mixture  of  toxin  and 

1  Ehrlich,    Die    Werthbemessung    des   Diphtherieheilserums. 
Klinisches  Jahrbuch,  1897. 


24  IMMUNE   SERA 

antitoxin,  would  require  the  addition  of  but  one 
fatal  dose  of  toxin  in  order  to  regularly  kill  the  test 
animal.  In  the  above  table,  however,  we  see  that 
this  difference  ranges  from  six  to  fifty  fatal  doses. 

Partial  Saturation  Method  -  -  Toxons,  Toxoids.  — 
Ehrlich  obtained  considerable  additional  informa- 
tion by  means  of  his  "  partial  saturation  "  method. 
Certain  experiments  had  led  him  to  believe  that  the 
original  antitoxin  on  which  he  had  based  his  "  unit  " 
determinations,  while  able  to  neutralize  100  fatal 
doses  (per  unit)  really  represented  200  "  binding 
units,"  and  that  the  toxic  bouillon  really  contained 
several  kinds  of  poisonous  substances  able  to  com- 
bine with  antitoxin. 

He  now  believes  that  the  diphtheria  bacilli  excrete 
at  least  two  such  poisons,  "  toxins  "  and  "  toxons ;  " 
that  these  very  quickly  decompose  to  a  greater  or 
less  extent  forming  various  "  toxoids." 

In  the  case  of  a  hypothetically  pure  toxin  Ehrlich 
believes  that  one  antitoxic  unit  would  correspond 
to  200  fatal  doses  or  200  binding  units.  If  the 
entire  amount  of  antitoxin,  i.e.  |§§  is  added  to 
the  amount  of  toxin  in  question,  the  result  will  be 
just  complete  neutralization.  If  the  toxin  is  entirely 
pure,  ^{jf  of  the  antitoxin  unit  would  neutralize  all 
but  ?hu  of  the  initial  toxicity  and  M&,  or  ?$%  or  ^A, 
etc.  of  the  antitoxin  added  would  permit  correspond- 
ing degrees  of  toxicity  to  be  demonstrated  through 
animal  inoculations.  It  was  found,  however,  that 
neutralization  according  to  this  simple  scale  did  not 


ANTITOXINS  25 

take  place.  The  results  were  complicated  andEhrlich 
found  it  convenient  to  express  them  graphically  in 
the  form  of  the  so-called  "toxin  spectra.  "  Without 


0     10   20    30    40 


Toxon 


70    80   90  100 


150 


200 


FlG.    2. 

going  much  deeper  into  the  subject  the  point  maybe 
illustrated  by  the  appended  diagrams  or  "  spectra." 

Fig.  2  shows  the  simplest  conceivable  diphtheria 
poison.  In  this  case  the  following  values  would 
be  obtained. 

#cc  poison  (100  fatal  doses)  +  f  §§  antitoxin 
units  =  o,  i.e.  absolutely  neutral. 

#cc  poison  +  iffft  =  Free  toxon. 

#cc  poison  +  *£{}  =  Free  toxon. 

That  is  to  say,  if  the  proportion  of  antitoxin  added 
was  Mti  of  the  amount  required  for  complete 
neutralization,  it  would  be  found  that  the  poison 
thus  uncombined  was  much  less,  and  differently 
toxic  than  a  corresponding  amount  of  the  original 
toxin.  It  was  found  that  these  fractions  possessed 
a  rather  constant  though  low  degree  of  toxicity 
with  characteristic  action.  This  consisted  in  the 
production  of  some  local  oedema,  followed  by  a 
long  incubation  period,  and  finally  the  develop- 
ment of  cachexia  and  paralysis.  Ehrlich  believes 


26 


IMMUNE   SERA 


that  this  action  is  due  to  a  separate  poison  excreted 
by  the  diphtheria  bacillus  which  he  calls  a  toxon. 

If  we  continue  with  the  above  poison  we  shall 
obtain  these  values: 

#cc  poison  +  ^  =  Toxin  action    (i  fatal  dose). 

#cc  poison  +  sVu  =30  fatal  doses. 

xcc  poison  +  sVk  =  90  fatal  doses,  etc. 

That  is  to  say,  if  we  add  only  $fo  units  antitoxin, 
i.e.  sitf  unit  less  than  in  the  JH  mixture,  we  find 
that  one  fatal  dose  is  set  free.  This  relation  would 
exist  right  to  the  end.  The  fact  that  in  this  experi- 
ment the  toxins  are  liberated  after  the  toxons, 
shows  that  the  toxons  have  less  affinity  for  the  anti- 
toxin than  have  the  toxins. 

As  a  matter  of  fact,  however,  conditions  are  prob- 
ably never  as  simple  as  this.  In  the  process  of 
toxin  formation  a  double  action  is  always  going 
on  —  that  of  toxin  and  toxon  production,  and  that 
of  their  decomposition.  As  was  pointed  out  on 
a  previous  page  the  poisons  quickly  change  into 
non-poisonous  toxoids,  and  these  substances  are 
still  able  to  bind  antitoxin. 

This  is  shown  in  the  following  "  spectrum." 


Protoxoid 


0     10    20    30    40    50    60 


1GO 

FIG.  3. 


Toxon 


150  160 


ANTITOXINS 

Here  we  would  obtain  the  following  figures: 


27 


#cc  poison  -f 
lutely  neutral. 

#cc  poison  + 

#cc  poison  + 

#cc  poison  -f 

oo™  poison  + 

occc  poison  + 


antitoxin     unit  =  o,   i.e.     abso- 


= Toxon  free. 
=  Toxon  free. 

=  Toxin  free  (i  fatal  dose.) 
-  Toxin  free  (60  fatal  doses.  ) 
=  Toxin  free  (100  fatal  doses.) 
Now  we  come  to  the  non-poisonous  "prototoxoids"  : 
#cc  +  inm  =  Toxin  free(ioo  fatal  doses.) 
#cc  +  *o°o   =  Toxin  free  (100  fatal  doses.) 
xcc  +  sio   =  Toxin  free  (100  fatal  doses.) 
We    see   here    that    after  we   have   reduced    the 
antitoxin   to  ^  no  further  increase   of  toxicity  is 
brought  about  by  any  further  reductions.     Ehrlich 
calls   these   toxoids    "  prototoxoids  "   because   they 
have  such  a  high  affinity  for  the  antitoxin.     But 
there  are  apparently  still  other  toxoids,  as  is  shown 
by  the  following  spectrum: 


Protoxoid 


Syntoxoid 


Toxon 


'    100    '  10    '  '200 

FIG.  4. 

Here  we  would  obtain  values  as  follows: 
xcc  poison  -f-  f  $%  =  o,  i.e.  absolutely  neutral. 
xce  poison  -f  iol  =  Toxon. 


28  IMMUNE   SERA 

xcc  poison  4-  M<!  =  Toxin  free  (i  fatal  dose). 

occc  poison  +  Mf  =  Toxin  free  (  2  fatal  doses.) 

xcc  poison  4-  ^B§  =  Toxon  free  (30  fatal  doses.) 

Here  we  find  that  in  the  middle  part  of  the 
"  spectrum  "  we  encounter  a  zone  in  which  each  ^"o 
antitoxin  unit  neutralizes  one  fatal  dose.  Ehrlich 
believes  that  this  part  of  the  mixture  consists  of 
equal  parts  of  syntoxoid  and  toxin — that  is  to 
say,  he  believes  there  are  also  toxoids  which 
have  the  same  degree  of  affinity  for  antitoxin 
that  this  toxin  has.  He  speaks  of  these  as  "  syn- 
toxoids." 

By  following  out  this  conception  of  the  toxin- 
antitoxin  combination,  Ehrlich  comes  to  the  con- 
clusion that  diphtheria  poison  is  a  very  complex 
substance,  containing  toxin,  toxon,  and  perhaps  still 
other  primary  secretion  products  in  addition  to  the 
various  secondary  modifications  of  these,  toxoids, 
toxonoids,  etc.  It  is  difficult  to  escape  the  feeling 
that  the  existence  of  some  of  these  hypothetical 
substances  is  more  apparent  than  real. 

Views  of  Arrhenius,  Bar  del  and  Others. — Bordet 
and  others  refuse  to  accept  Ehrlich 's  views  and 
the  whole  matter  is  still  unsettled.  Thus  the  exist- 
ence or  non-existence  of  toxons  has  excited  a  great 
deal  of  discussion  among  investigators. 

The  great  Swedish  chemist,  Arrhenius,  has  given 
much  attention  to  the  toxins;  and  has  applied 
the  principles  of  physical  chemistry  to  the  toxin- 


ANTITOXINS 


29 


antitoxin  reaction.  It  is,  of  course,  well  known 
that  a  solution  of  a  compound  such  as  sodium 
chloride  represents  not  only  NaCl  in  solution, 
but  also  sodium  ions  and  chlorine  ions.  There  is 
a  certain  amount  of  dissociation  going  on  hand  in 
hand  with  a  combination  of  the  two  components. 
The  degree  of  this  varies  with  the  temperature  and 
the  dilution  of  the  substances.  Arrhenius  believes 
that  the  same  process  goes  on  with  the  toxin- 
antitoxin  combination  and  that  such  more  or  less 
dissociated  compounds  give  rise  'to  the  effects 
Ehrlich  ascribes  to  the  toxon.  There  is,  however, 
no  direct  evidence  that  the  combination  of  toxin- 
antitoxin  is  reversible.  It  is  true  that  Morgenroth 
has  been  able  to  dissociate  the  two  components  of 
a  neutral  mixture  of  cobra  venom  and  its  antitoxin. 
But  even  here  we  are  not  dealing  with  a  reversible 
reaction,  for  it  requires  certain  manipulations  to 
disrupt  the  neutral  combination.  In  their  work 
on  the  toxin  of  symptomatic  anthrax,  Grassberger 
and  Schattenfroh  found  that  different  mixtures 
were  obtained,  depending  on  whether  they  mixed 
the  toxin  and  antitoxin  after  diluting  them,  or 
diluted  the  toxin-antitoxin  mixture.  This  fact 
is  not  in  favor  of  Arrhenius'  theory,  for  according 
to  that,  the  same  state  of  equilibrium  should  exist 
in  both  instances  owing  to  reversibility,  and  the 
same  fraction  of  the  toxin  of  necessity  remain 
free. 


30  IMMUNE  SERA 

Bordet  *  believes  that  the  neutralization  of  toxin 
by  antitoxin  is  an  adsorption  phenomenon,  and 
compares  it  with  the  process  of  dyeing.  The 
molecules  of  the  toxin  would  "  stain  "  more  or  less 
deeply  by  the  antitoxin  molecule,  and  the  com- 
plexes that  result  in  the  various  instances  would 
be  less  toxic  in  proportion  as  they  contained  more 
antitoxin  and  less  toxin.  If  a  large  piece  of  filter 
paper  is  placed  in  a  certain  volume  of  sufficiently 
diluted  dye,  it  takes  a  uniform  shade  of  intensity; 
if,  on  the  other  hand,  the  same  sized  piece  of  paper 
is  cut  in  pieces  and  added  in  fragments,  the  first 
pieces  are  stained  deeply,  and  the  last  find  no  color 
left.  In  the  same  way,  on  adding  toxin  to  antitoxin 
in  divided  doses,  the  last  portions  of  the  poison 
cannot  be  neutralized,  as  the  first  are  supersatu- 
rated with  antitoxin.  When  the  entire  mixture 
is  made  at  once,  on  the  contrary,  the  antitoxin  is 
spread  all  over  the  toxin  molecules  and  a  complex 
is  obtained  which  contains  an  even  proportion  of 
the  antidote,  and  which,  consequently,  is  not  as 
fatal  as  even  a  small  dose  of  free  toxin.  The  action 
which  Ehrlich  therefore  ascribes  to  toxons,  Bordet 
refers  to  toxin  which  is  partially  saturated  with 
antitoxin.  Bordet  also  cites  the  researches  of 
Grassberger  and  Schattenfroh  on  the  toxin  of 
symptomatic  anthrax.  The  toxic  fluid  which  these 

1  Bordet-Gay,  Collected  Studies  in  Immunity,  Wiley  &  Sons, 
1909. 


ANTITOXINS  3I 

authors  employed  contains  only  a  single  poison: 
there  is  no  reason  for  assuming  the  existence  of 
toxoids,  inasmuch  as  the  toxic  power  of  the  poison 
is  constantly  parallel  to  its  neutralizing  power  for 
antitoxin.  On  mixing  a  certain  dose  of  the  toxin 
either  with  little  or  with  much  antitoxin,  complexes 
of  toxin-antitoxin  were  obtained  which  varied 
in  their  reaction  to  heat.  Moreover,  these  authors 
found  that  their  poison  absorbs  much  more  anti- 
toxin than  is  necessary  to  destroy  its  entire  toxicity, 
and  forms  a  stable  compound  with  it.  Bordet's  con- 
ception of  the  toxin-antitoxin  reaction  thus  seems 
to  be  very  simple.  The  main  difficulty  which  it 
encounters  is  the  strict  specificity  of  the  combi- 
nation. However,  recent  investigations  make  it 
probable  that  the  affinity  of  adsorption  is  similar 
to  a  true  chemical  affinity,  in  that  both  are  elective. 
It  is  possible,  therefore,  that  the  existence  of  strict 
specificity  may  still  be  found  entirely  compatible 
with  the  adsorption  theory. 


AGGLUTININS 

The  Agglutination  Phenomenon.  —  We  have  just 
seen  that  pathogenic  bacteria  may  be  divided  into 
those  which  produce  extracellular  toxins  in  culture 
media,  and  those  which  do  not.  Against  the 
former  the  organism  defends  itself  by  the  production 
of  antitoxins ;  against  the  latter  it  produces  a  variety 
of  antibodies :  —  bacteriolysins,  agglutinins,  precipi- 
tins,  opsonins  and  possibly  others. 

The  agglutinins  can  be  observed  either  in  a  test- 
tube  or  in  a  microscopical  preparation.  For  example, 
if  typhoid  or  cholera  immune  sera  are  added  respec- 
tively to  a  24-hour  culture  of  typhoid  or  cholera 
bacilli,  and  the  mixture  placed  in  a  thermostat, 
the  following  phenomenon  will  be  noticed:  The 
bacteria  which  previously  clouded  the  bouillon 
uniformly,  clump  together  into  little  masses,  settle 
to  the  sides  of  the  test-tub  2  and  gradually  fall  to 
the  bottom  until  the  fluid  is  almost  entirely  clear. 
In  a  control  test,  on  the  contrary,  to  which  no  active 
serum  is  added,  the  fluid  remains  uniformly  cloudy. 
The  reaction  is  completed  in  twenty-four  hours 
at  the  most.  If  the  reaction  is  observed  in  a  hang- 
ing drop,  it  is  seen  that  the  addition  of  the  active 

serum  first  produces  an  increased  motility  of  the 

32 


AGGLUTIN1NS  33 

bacteria  which  lasts  a  short  time  and  is  followed 
by  a  gradual  formation  of  clumps.  One  gets  the 
impression  that  the  bacteria  are  dying  together. 
Frequently  one  sees  bacteria  which  have  recently 
joined  a  group  make  violent  motions  as  though  they 
were  attempting  to  tear  themselves  away;  then 
they  gradually  lose  their  motility  completely.  Even 
the  larger  groups  of  bacteria  may  exhibit  movement 
as  a  whole.  After  not  more  than  one  or  two  hours 
the  reaction  is  completed;  in  place  of  the  bacteria 
moving  quickly  across  the  field,  one  sees  one  or 
several  groups  of  absolutely  immobile  bacilli.  Now 
and  then  in  a  number  of  preparations  one  sees 
a  few  separate  bacteria  still  moving  about  among 
the  groups.  If  the  reaction  is  feeble,  either  because 
the  immune  serum  has  been  strongly  diluted  or 
because  it  contains  very  little  agglutinin,  the  groups 
are  small  and  one  finds  comparatively  many  iso- 
lated and  perhaps  also  moving  bacteria.  It  is 
essential  each  time  to  make  a  control  test  of  the 
same  bacterial  culture  withoiit  the  addition  of 
serum.  Under  some  circumstances  the  reaction 
proceeds  with  extraordinary  rapidity  so  that  the 
bacilli  are  clumped  almost  immediately.  By  the 
time  the  microscopical  slide  has  been  prepared 
and  brought  into  view  nothing  is  to  be  seen 
of  any  moving  or  isolated  bacteria,  and  only  by 
means  of  the  control  test  is  it  t  possible  to  tell 
whether  the  culture  possessed  normal  motility. 


34  IMMUXE  SERA 

We  are  not  yet  informed  as  to  the  nature  of  these 
phenomena.  A  number  of  theories  have  been  ad- 
vanced, into  which,  however,  we  cannot  here  enter. 

In  some  cases  the  agglutinins  are  active  even  in 
very  high  dilutions.  Thus  in  typhoid  patients 
and  typhoid  convalescents  a  distinct  agglutination 
has  been  observed  in  dilutions  of  i :  5000,  and  this 
action  persisted  for  years,  though  not,  of  course, 
in  the  same  degree.  Even  normal  blood-serum, 
when  undiluted,  often  produces  agglutination.  But 
the  above  specific  agglutinins,  which  do  not  exist 
beforehand,  being  formed  only  in  consequence  of 
an  infection,  are  characterized  by  this,  that  the 
agglutination  occurs  even  when  the  serum  is  diluted 
(at  least  i  :  30  to  i  :  50),  and,  furthermore,  that  after 
this  dilution  the  action  is  still  specific,  i.e.  cholera 
immune  serum  agglutinates  only  cholera  bacilli, 
typhoid  immune  serum  only  typhoid  bacilli,  etc. 
This  specificity,  however,  as  will  be  shown  later, 
is  not  always  absolute. 

Agglutinins  can  also  be  developed  against  red 
blood  cells  and  against  certain  protozoa  (trypan- 
osomes).  We  speak  of  iha  former  as  hamag- 
glutinins.  Analogous  to  the  hsemolytic  action  or 
normal  serum  on  the  red  cells  of  certain  other 
species,  we  find  that  normal  serum  is  able  to 
agglutinate  the  red  cells  of  many  species  and  bac- 
teria. For  example,  normal  goat  serum  aggluti- 
nates the  red  cells  of  man,  pigeon,  and  rabbit; 


AGGLUTININS  35 

normal    rabbit    serum    agglutinates    typhoid    and 
cholera  bacilli. 

Of  practical  interest  is  the  fact  that  normal  serum 
may  agglutinate  the  red  blood  cells  of  another  indi- 
vidual of  the  same  species.  Following  Ehrlich's  nomen- 
clature, we  speak  of  this  as  is o agglutination.  The 
subject  has  been  studied  by  a  number  of  investigators, 
and  mostly  in  human  blood.  According  to  the  extensive 
investigations  of  Moss,  isoagglutinins  occur  in  the  serum 
of  about  90%  of  adult  human  beings.  Landsteiner 
divided  the  individuals  into  three  groups,  namely : 

Group  i. — The  corpuscles  are  not  agglutinated  by 
sera  of  the  other  two  groups,  while  the  sera  agglu- 
tinate the  corpuscles  of  both  groups. 

Group  2. — The  corpuscles  are  agglutinated  by  the 
sera  of  the  other  two  groups,  while  the  sera  agglutinate 
the  corpuscles  of  Group  3,  but  not  of  Group  i. 

Group  3. — The  corpuscles  are  agglutinated  by  the 
other  two  sera,  and  the  sera  agglutinate  the  corpuscles 
of  Group  2,  but  not  of  Group  i. 

An  examination  of  this  grouping  shows  that  in  no 
case  is  there  an  agglutination  of  erythrocytes  by  their 
own  serum,  in  other  words  these  are  isoagglutinins  but 
not  autoagglutinins.  A  somewhat  different  classification 
was  made  by  Jansky,  and  independently  of  him  also  by 
Moss.  Both  these  authors  find  it  necessary  to  establish 
four  groups  in  order  to  embrace  all  the  cases  met  with. 

Gay  calls  attention  to  the  fact  that  the  clumping  of 
erythrocytes  by  serum  is  not  necessarily  due  to  the  pres- 
ence of  an  agglutinin  at  all,  but  may  be  due  to  variations 
in  the  molecular  concentration  of  the  serum  constituents 
or  of  the  constituents  of  the  blood  cells.  Whatever  finally 
proves  to  be  the  correct  explanation,  it  is  obvious  that 
the  occurrence  of  isoagglutination  in  human  blood  might 


36  IMMUNE   SERA 

possibly  prove  disastrous  in  human  homologous  trans- 
fusion. For  this  reason  it  is  now  common  practice  to 
always  precede  homologous  transfusion  by  an  examina- 
tion of  the  blood  of  both  donor  and  recipient.  A  brief 
outline  of  these  tests  is  given  in  the  appendix. 

Purpose  of  Agglutination.  —  It  is  not  yet  clear 
what  the  purpose,  if  any,  of  ths  agglutinating 
function  is.  Gruber,  the  first  to  thoroughly  study 
and  appreciate  the  bacterial  agglutinins,  assumes 
that  the  process  injures  the  affected  cell,  preparing 
it  for  solution  and  destruction.  After  numerous 
experiments  I  have  not  been  able  to  convince 
myself  of  any  damaging  influence  of  the  agglutinins 
on  the  affected  cell,  be  this  blood  cell  or  bacterium, 
and  the  observations  of  other  authors  confirm  this 
opinion.  Agglutinated  bacteria  are  capable  of 
living  and  of  reproduction,  and  agglutinated  red 
blood  cells  are  no  more  fragile  or  easier  to  destroy 
than  normal,  non-agglutinated  cells.  Neither  can 
anything  be  discovered  microscopically  which  would 
indicate  any  injury  to  their  structure. 

One  thing  is  certain:  that  the  agglutinins  are  in 
no  way  related  to  the  lysins  found  in  serum,  and 
so  of  course  are  not  identical  with  these.  The 
simultaneous  occurrence  in  a  serum  of  immune 
bodies,  interbodies,  complements,  and  agglutinins 
is  an  entirely  independent  phenomenon  which  is 
in  no  way  regular.  There  are  sera  which  dissolve 
certain  cells  without  agglutinating  them,  and  others 
which  agglutinate  cells  without  dissolving  them. 


AGGLUTININS  37 

Historical Serum  diagnosis  by  means    of    the 

agglutinins  was  introduced  chiefly  through  the 
labors  of  Gruber  and  Widal.  The  studies  under- 
taken by  Gruber  and  his  pupil  Durham  began  as 
early  as  1894.  At  the  Congress  for  Internal  Medi- 
cine in  1896  l  Gruber  first  announced  that  he  had 
discovered  the  reaction  in  typhoid  convalescents, 
and  asked  that  his  observations  be  verified  if  pos- 
sible. Soon  after  this  Pfeiffer  and  his  co-workers 
published  a  study  which  confirmed  Gruber's  results.2 
The  significance  of  the  reaction  as  a  diagnostic 
help  was  unquestionably  first  pointed  out  by  Widal,3 
who  showed  that  the  reaction  appears  at  a  relatively 
early  period  of  the  disease,  and  may  therefore  be 
employed  as  a  diagnostic  measure.  We  must  not 
omit  to  state  that  Griinbaum4  in  March,  1896,  several 
months  before  Widal's  publication,  had  also  grasped 
the  significance  of  the  reaction  as  a  diagnostic 
measure.  Owing  to  insufficient  clinical  material 
his  publication  did  not  appear  until  some  time  after 
Widal's.  Hence,  in  acknowledgment  of  the  labors 
of  the  two  authors  most  concerned  in  the  discovery 
and  introduction  of  this  reaction,  we  now  speak 

1  Transactions  of  the  Congress,  edited  by  E.  von  Leyden  and 
R.  Pfeiffer,  Wiesbaden,  1896. 

2  Pfeiffer  and   Kolle,  Deutsche    med.    Wochenschrift,   1896, 
No.  12. 

3  Widal,  Bulletin  de  la  soc.  mdd.  des  hop.,  June  26,  1896. 

4  Grunbaum,  Lancet,  Sept.  19,  1896;  Muench.  med.  Wochen- 
schrift, 1897,  No.   13;  Blood  and  the  identification  of  bacterial 
species,  Science  Progress,  Vol.  I,  No.  5,  1897. 


38  IMMUNE   SERA 

of  it  as  the  "Gruber-Widal  reaction,"  whereas  in 
the  beginning  only  the  term  "  Widal  reaction  " 
was  used. 

The  manner  in  which  the  reaction  proceeds  in 
microscopical  preparations  as  well  as  when  mac- 
roscopically  observed  has  been  described  above 
(page  32).  Nowadays  the  microscopic  method  is 
given  the  preference  x  because  in  many  cases  it  is 
distinct  when  the  macroscopic  reaction  fails;  and 
further  because  the  former  yields  distinct  results 
within  an  hour  at  the  most,  whereas  in  many  cases 
twenty-four  hours  are  required  for  the  macroscopic 
test. 

Pfaundler's  Reaction  (Thread  Reaction).  —  It 
may  be  well  at  this  point  to  call  attention  to  a 
peculiar  reaction  described  by  Pfaundler2  in  1896. 
This  author  showed  that  certain  bacteria,  though 
they  might  not  be  agglutinated  by  a  given  serum, 
would  often,  when  they  were  grown  therein,  develop 
in  the  form  of  long  threads  more  or  less  interlaced. 
This  occurred  only  in  the  specific  serum  and  wa£ 
absent  in  the  normal  serum.  Most  authorities 
regard  the  thread  reaction  as  a  manifestation  of 
agglutinins.  According  to  Metchnikoff  this  reaction 
sometimes  gives  more  information  concerning  a 
serum  than  does  the  ordinary  agglutination  test. 

1  This  applies  to  typhoid ;  in  other  diseases  the  macroscopic 
method  is  sometimes  preferable, 

'*  Pfaundler,  Centralblatt  Bacteriologie,  Vol.  xix,  1896. 


AGGLUTININS 


39 


Nature  of  the  Agglutinins,  and  of  the  Agglutina- 
tion Reaction. — The  agglutinins  are  fairly  resistant 
substances  which  withstand  heating  to  60°  C.,  and 
lose  their  power  only  on  heating  to  65°  C.  It  is  pos- 
sible, therefore,  to  make  a  serum  bacteriolytically  in- 
active by  heating  to  5  5°  C.,  and  still  preserve  its  agglu- 
tinating power.  It  has  been  found  that  agglutinins 
when  heated  may  keep  the  property  of  uniting 
with  bacteria,  although  they  lose  the  property  of 
agglutinating  them.  To  explain  this  fact,  Ehrlich 
supposes  that  agglutinins  possess  two  groups,  a 
haptophore  group,  effecting  the  specific  union  with 
the  cell,  and  an  ergophore  group,  which  effects  the 
clumping.  He  supposes  further  that  under  the  con- 
ditions mentioned  the  agglutinin  loses  its  aggluti- 
nating group  but  keeps  its  combining  group.  Such 
a  modified  agglutinin  Ehrlich  calls  an  agglutinoid, 
just  as  toxins  which  have  lost  their  toxophore 
groups  are  called  toxoids.  The  nature  of  agglutinoid, 
however,  is  still  very  obscure.  In  fact,  as  we  shall 
presently  see,  the  opponents  of  the  Ehrlich  school 
refuse  to  believe  in  the  existence  of  agglutinoids. 
It  has  occasionally  been  observed  that  agglutination 
is  absent  in  concentrated  serum,  and  present  in  dilute 
serum.  This  zone  of  no  agglutination,  preceding  that 
of  agglutination,  is  often  spoken  of  as  the  pro  zone 
and  was  first  described  by  Eisenberg  and  Volk.  Ac- 
cording to  Ehrlich.  it  is  due  to  the  presence  in  the 
serum  of  agglutinoids.  These  are  assumed  to  possess 
higher  affinity  for  the  bacteria  than  do  the  agglutinins 


4o  IMMUNE  SERA 

and  so  prevent  the  latter  from  acting  on  the  bacteria. 
Since,  however,  the  agglutinins  are  usually  far  more 
abundant  than  the  agglutinoids,  dilution  of  the  serum 
dilutes  the  latter  to  practically  nothing,  thus  allow- 
ing the  agglutinins  to  combine  with  the  bacteria. 

Ehrlich's  conception  of  the  structure  of  the  agglu- 
tinin  molecule  and  his  views  on  the  nature  of  the 
agglutination  reaction  have  been  sharply  combated. 

Elser  very  properly  points  out  that  not  enough 
attention  has  been  paid  to  the  effect  of  heat  on  serum, 
and  that  alterations  in  the  physical  characters  of  the 
serum  may  be  sufficient  to  account  for  phenomena 
heretofore  ascribed  to  chemical  changes.  Among 
other  things  he  cites  the  effect  of  heat  on  horse  serum ; 
heating  produces  a  marked  increase  in  the  viscosity 
of  the  serum.  It  is  obvious,  therefore,  when  heated 
sera  are  used  in  agglutination  experiments,  that  this 
purely  physical  characteristic  exerts  a  profound  in- 
fluence on  the  result  of  the  reaction.  Differences  in 
the  behavior  of  an  agglutinating  serum  before  and 
after  heating  must  therefore  be  interpreted  with  great 
caution,  and  must  not  at  once  be  taken  to  indicate 
the  chemical  alteration  of  the  agglutinin  complex. 

Bordet,  for  example,  cites  an  interesting  experi- 
ment of  Gengou.  An  aqueous  solution  of  agar, 
so  diluted  as  to  be  only  slightly  viscous  at  room 
temperature,  agglutinates  barium  sulphate  sus- 
pended in  water.  Heating  such  a  solution  destroys 
this  property  without  affecting  the  adsorbing 
property;  under  these  conditions  it  produces  the 


AGGLUTININS  4! 

opposite  effect,  namely,  disseminates  the  particles 
of  barium  and  gives  a  milky  appearance  to  the 
fluid.  Can  we,  says  Bordet,  claim  that  by  heating 
this  solution  we  have  caused  it  to  lose  its  agglu- 
tinating group?  Bordet  agrees  with  Forges,  who 
believes  that  the  hypothesis  of  such  a  group  in  the 
antibody  molecule  has  no  foundation.  Forges 
found,  on  studying  the  effect  of  heat  on  the  agglu- 
tinating power  of  the  albuminous  substances  of 
serum  for  mastic  emulsions,  that  he  could  obtain 
results  entirely  similar  to  those  that  have  been 
noted  for  agglutinins.  Bordet  insists  that  we  have 
no  right  to  localize  the  cause  of  agglutination  in  a 
molecule  of  the  antibody  rather  than  in  one  of  the 
antigen.  The  hypothesis  of  a  functional  group  in 
the  molecule  of  the  .agglutinin,  he  says,  is  all  the 
more  doubtful,  inasmuch  as  it  is  not  the  only  sub- 
stance which  can  render  bacteria  sensitive  to  the 
flocculating  action  of  salts.  Bacteria  that  have 
adsorbed  iron,  uranium,  or  aluminium  compounds 
are  subsequently  flocculable  by  salts,  and  silicic 
acid  is  similar  in  its  action.  According  to  Bordet, 
the  essential  phenomenon  with  agglutination,  as 
with  other  active  substances  in  sera,  is  its  union 
with  the  antigen;  as  far  as  the  agglutination  itself, 
which  follows  this  union,  is  concerned,  it  is  only  a 
secondary  phenomenon  on  which  we  cannot  depend 
in  considering  agglutinins  as  functionally  different 
in  molecular  structure  from  the  other  antibodies. 
The  influence  of  salts  upon  agglutination  is  in 


4 2  IMMUNE  SERA 

a  sense  comparable  to  their  action  upon  the  pre- 
cipitins.  Joos  found  that  antityphoid  serum  did 
not  agglutinate  typhoid  bacilli  in  the  absence  of 
salts.  For  agglutination  to  take  place  he  considers 
it  as  necessary  as  the  agglutinin  and  agglutinable 
substance.  He  believes  that  salts  play  an  active 
part  in  the  process.  Bordet,  on  the  other  hand, 
believes  that  the  absence  of  salts  offers  only  a 
physical  impediment  to  agglutination.  Friedberger 
does  not  consider  that  the  salts  act  chemically,  for  he 
found  that  agglutination  took  place  in  the  presence 
of  grape  sugar,  asparigin,  etc.,  in  the  place  of  salts. 
In  view  of  the  fact  that  the  protoplasm  of  the 
body  and  the  albuminous  constituents  of  serum 
have  a  close  relationship  to,  or  really  are,  colloids, 
investigators  have  studied  certain  reactions  which 
occur  among  the  colloids  with  the  expectation 
that  these  would  throw  some  light  on  the  reactions 
of  protoplasm  and  of  serums.1  Colloids  diffuse 
very  slowly  and  exert  little  or  no  osmotic  pressure, 
supposedly  because  of  the  large  size  of  the  particles. 
They  do  not  conduct  electricity,  but  the  particles 
react  to  the  electric  current  by  alterations  in  the 
direction  of  their  motion  (i.e.,  toward  the  positive 
or  the  negative  pole)  and,  moreover,  carry  electric 
charges  themselves.  The  features  of  colloids  which 
bring  them  into  relation  with  the  subject  in  hand 
are  their  coagulable  nature  in  certain  instances  and 
the  fact  that  their  particles  may  be  agglutinated 

1  This  subject  is  well  presented  in  :     Pauli- Fischer,  Physical 
Chemisty  in  the  Service  of  Medicine.     Wiley  &  Sons,  N.  Y. 


AGGLUTININS  43 

or  precipitated  by  the  addition  of  minute  amounts 
of  salts  (electrolytes).  This  of  course  is  entirely 
analogous  to  the  need  of  salts  in  the  agglutination 
of  bacteria  by  sera.  In  the  latter  reaction  the 
agglutinins  carry  a  positive,  the  bacteria  a  negative 
charge.  The  resulting  combination,  therefore,  does 
not  precipitate  from  the  menstruum  supposedly 
because  there  is  still  sufficient  difference  in  the 
electric  potential.  When  salts  are  present  the 
kations  so  alter  the  electric  conditions,  of  the  colloi- 
dal particles,  i.e.,  of  the  agglutinin-bacterium  com- 
bination, that  their  surface  tension  is  increased. 
In  order  to  overcome  this  the  particles  get  together, 
presenting  in  a  clump  less  surface  tension  than  if 
they  remained  as  individual  particles.  Some  experi- 
ments by  Field  indicate  that  the  pro  zone  may  be 
explained  on  the  assumption  that  the  bacteria  and 
agglutinins  behave  as  colloids.  It  has  already 
been  stated  that  the  union  of  agglutinin  and  bac- 
terium does  not  precipitate  because,  possibly,  there 
is  still  sufficient  electric  potential;  the  combination 
carries  a  negative  charge.  Field  believes  that  with 
very  large  amounts  of  agglutinin  (as  in  the  pro 
zone)  the  bacteria  load  themselves  with  so  much 
agglutinin  that  the  combination  now  carries  a  con- 
siderable positive  charge.  The  surface  tension  there- 
fore is  not  sufficient  to  cause  a  clumping  to  occur. 
Naturally,  the  presence  of  salts  does  not  alter  the  con- 
dition, as  the  kations  also  carry  a  positive  charge. 
Group  Agglutinins. —  For  some  time  after  their 


44 


IMMUNE  SERA 


discovery  the  agglutinins  were  regarded  as  strictly 
specific,  i.e.,  a  serum  derived,  for  example,  from  a 
typhoid  infection  would  agglutinate  only  typhoid 
bacilli  and  no  others.  After  a  time,  however,  it  was 
found  that  such  a  serum  would  frequently  aggluti- 
nate somewhat  related  organisms,  though  not, 
usually,  to  so  high  a  degree.  In  other  words,  while 
agglutinins  may  be  nearly,  if  not  quite,  specific  in 
their  action,  a  serum  which  produces  agglutination 
may  be  far  from  being  so. 

The  following  examples  will  illustrate  the  point. 
In  a  case  of  infection  with  paratyphoid  bacilli, 
type  B,  the  bacilli  of  the  infecting  type  B  were 
agglutinated  1:5700;  typhoid  bacilli,  however,  only 
1:120,  while  paratyphoid  bacilli  type  A  were  not 
agglutinated  at  all.  In  a  case  of  typhoid  infection 
an  agglutination  with  a  dilution  of  i :  40  was  obtained 
for  paratyphoid  type  B,  while  typhoid  bacilli  were 
agglutinated  in  a  dilution  of  i :  300  and  over.  As  a 
rule  the  agglutination  with  the  infecting  agent  is  by 
far  the  strongest,  i.e.  it  proceeds  even  in  high  dilu- 
tions, whereas  other  bacteria  require  a  stronger 
concentration. 

This  phenomenon  is  known  as  group  agglutina- 
tion. The  bacteria  which  are  agglutinated  by  one 
and  the  same  serum  need  not  necessarily  be  related, 
although  usually  this  is  the  case.  Conversely, 
microorganisms  which,  because  of  their  morpho- 
logical or  other  biological  characteristics,  are  re- 
garded as  entirely  identical  or  nearly  so,  are  sharply 


AGGLUTININS 


45 


differentiated  by  means  of  their  agglutination. 
Because  of  this  lack  of  absolute  specificity  the  serum 
diagnosis  of  infection  or  the  identification  of  bac- 
teria by  means  of  agglutination  tests,  has  value 
only  when  very  carefully  tested.  We  have  said 
above  that  while  agglutinins  are  specific,  a  serum 
which  produces  agglutination  may  be  far  from 
being  so.  The  reason  for  this  is  that  the  serum 
may  contain  several  agglutinins.  In  fact,  when 
immunizing  an  animal  with  a  particular  bacterium 
both  specific  and  group  agglutinins  are  produced. 
This  will  perhaps  be  made  clearer  by  reference  to 
the  following  diagram.  We  assume  that  the  typhoid 

A  B       C  B  ft  E       F 


Typhoid  Bacillus 


Colon  Bacillus 


Dysentery  Bacillus 
FIG.  5. 

bacillus  possesses  considerable  protoplasm  A,  which 
is  specific  for  the  typhoid  bacillus;  that  it  possesses 
also  certain  protoplasm  B,  which  is  common  to  it, 
and  to  the  colon  bacillus;  and  some  protoplasm 
C,  common  perhaps  to  some  other  bacterium.  In 
the  case  of  the  colon  bacillus,  protoplasm  D  is 
specific,  i.e.,  possessed  only  by  this  bacillus,  while 
B  is  common  to  it  and  the  typhoid  bacillus,  and  E 
common  to  colon  and  dysentery  bacilli.  By  immu- 
nization with  the  typhoid  bacillus  we  would  obtain 


4  6  IMMUNE   SERA 

a  serum  containing  agglutinins  against  protoplasm 
A,  B,  and  C.  By  virtue  of  this  the  serum  \vould  exert 
some  agglutinating  power  also  on  colon  bacilli. 

Absorption  Method  for  Differentiating  between 
a  Mixed  and  a  Single  Infection  and  for  Identifying 
Bacteria.  —  In  1902  Castellani  called  attention  to 
a  procedure  which  consists  in  saturating  the  diluted 
immune  serum  with  successive  quantities  of  the 
bacteria  most  strongly  agglutinated  until  the  agglu- 
tinating power  for  these  is  zero.  After  centrifug- 
ing,  the  clear  fluid  is  tested  on  the  second  variety 
of  bacteria,  and  from  this  one  learns  whether  mixed 
or  single  infection  was  present.  According  to  Castel- 
lani, if  the  serum  of  an  animal  immunized  against  a 
certain  microorganism  is  saturated  with  that  organ- 
ism, the  serum  will  lose  its  agglutinating  power  not 
only  for  that  organism,  but  also  for  all  other  varieties 
that  it  formerly  acted  on.  Saturated  with  the  others, 
its  action  upon  the  first  is  reduced  little  or  none  at  all. 
The  serum  of  an  animal  immunized  against  two 
microorganisms  A  and  B.  loses  its  agglutination 
when  saturated  with  A,  only  for  A.  Saturated  with 
A  and  B  it  loses  agglutinating  power  for  both. 

The  absorption  test  is  extensively  used  in  the 
identification  of  bacteria,  but  it  must  be  used  with 
caution,  as  its  interpretation  is  open  to  error.  Refer- 
ring to  the  figure  illustrating  specific  and  group 
agglutinins,  let  us  assume  we  have  obtained  a 
specific  typhoid  serum  by  immunization  with  typhoid 
bacilli.  By  virtue  of  the  common  agglutinin,  this 


AGGLUTININS 


47 


serum  will  act  also  on  colon  bacilli.  On  extracting 
such  a  serum  with  typhoid  bacilli,  all  the  aggluti- 
nating power  would  be  lost,  that  for  typhoid  bacilli 
as  well  as  that  for  colon.  On  extracting  the  serum 
with  colon  bacilli,  we  would  remove  the  aggluti- 
nating power  for  these  bacilli,  but  leave  the  specific 
agglutinating  power  for  typhoid  bacilli.  If  we 
extracted  the  serum  with  a  culture  suspected  to  be 
typhoid  bacilli,  and  found  after  extraction  that 
the  serum  no  longer  agglutinated  known  typhoid 
bacilli,  we  could  conclude  that  the  suspected  culture 
was  also  one  of  typhoid  bacilli. 

Formation  of  the  Agglutinins  According  to  the 
Side-Chain  Theory  —  Receptors  of  First,  Second  and 
Third  Order.  —  Ehrlich's  theory  as  outlined  in  the 
preceding  chapter  offers  a  ready  explanation  for  the 
development  of  these  bodies.  Certain  peculiarities 
of  the  agglutinins  require  merely  a  slight  elabora- 
tion of  detail  in  order  to  be  clearly  understood. 
According  to  Ehrlich  the  prime  function  of  the  side 
chains  of  a  cell  is  to  provide  for  the  nutrition  of  the 
cell.  Obviously  the  simplest  mechanism  for  this 
purpose  will  be  a  side  chain  which  merely  anchors 
the  food  molecule,  leaving  the  digestion  entirely  to 
the  cell  proper.  This  type  of  receptor  suffices  for 
comparatively  small  molecules  such  as  those  of  the 
toxins,  for  these  are,  after  all,  but  the  products  of 
cellular  activity.  When  the  protoplasm  of  the 
bacterial  cell  itself,  however,  is  to  serve  as  food  for 
the  animal  cell  the  latter  needs  more  than  a  mere 


4g  IMMUNE   SERA 

anchoring  group,  it  needs  also  an  active  group 
which  can  in  some  way  act  on  the  huge  food  par- 
ticle and  make  it  more  readily  assimilable.  Such 
receptors  then  possess  two  groups,  a  haptophore 
group  and  another  functional  group  acting  on  the 
food  particle  thus  anchored.  Ehrlich  calls  these 
his  "  receptors  of  the  second  order,"  and  places  in 
this  class  the  agglutinins  and  the  precipitins.  The 
same  action  can  perhaps  be  more  economically 
brought  about  by  having  these  receptors,  in  addi- 
tion to  their  specific  haptophore  group,  possess  the 
means  by  which  the  action  of  a  ferment-like  sub- 
stance can  be  brought  to  bear  on  the  anchored 
food  particle.  Such  a  receptor  would  then  possess 
two  haptophore  groups,  one  for  the  food  particle, 
the  other  for  the  ferment-like  substance.  These 
are  Ehrlich's  "  receptors  of  the  third  order  "  and 
will  be  discussed  in  the  next  chapter.  Confining 
ourselves  for  the  present  to  the  agglutinins  we  find 
that  the  existence  of  the  two  groups  (haptophore 
and  agglutinating)  has  experimental  confirma- 
tion. We  have  seen  that  an  agglutinin  may  be 
changed  by  the  action,  for  instance,  of  acids,  so  that 
it  will  no  longer  possess  any  agglutinating  action, 
but  will  still  combine  with  the  bacteria.  Once  the 
agglutinating  power  is  lost  it  cannot  be  restored, 
in  which  respect  the  agglutinins  differ  from  the 
bacteriolysins. 


FIG.   6. — THE    VARIOUS    TYPES    OF     RECEPTORS    ACCORDING    TO 

EHRLICH. 

I.  Receptors  of  the  First  Order.  —  This   type  is   pictured    in  a.     The 

portion  e  represents  the  haptophore  group,  whilst  b  represents  a 
toxin  molecule,  which  possesses  a  haptophore  group  c  and  a 
toxophore  group  d.  This  represents  the  union  of  toxin  and 
antitoxin,  or  ferment  and  antiferment,  the  union  between  anti- 
body and  the  toxin  or  ferment  being  direct. 

II.  Receptors  of  the  Second  Order  are  pictured  in  c.      Here  e  represents 

the  haptophore  group,  and  d  the  zymophore  group  of  the 
receptor,  f  being  the  food  molecule  with  which  this  receptor 
combines.  Such  receptors  are  possessed  by  agglutinins  and 
precipitins.  It  is  to  be  noted  that  the  zymophore  group  is  an 
integral  part  of  the  receptor. 

III.  Receptors  of  the  Third  Order  are    pictured   in    III,  e  being  the 

haptophore  group  and  g  the  complementophile  group  of  the 
receptor.  The  complement  k  possesses  a  haptophore  group  h 
and  zymotoxic  group  z;  whilst  f  represents  the  food  molecule 
which  has  become  linked  to  the  receptor.  Such  receptors  are 
found  in  hsemolysins,  bacteriolysins,  and  other  cytolysins,  the 
union  with  these  cellular  elements  being  effected  by  the  ambo- 
ceptor  (a  thrust-off  receptor  of  this  order).  It  is  to  be  noted 
that  the  digesting  body,  the  complement,  is  distinct  from  the 
receptor,  a  point  in  which  these  receptors  therefore  differ  from 
those  of  the  preceding  order. 


BACTERIOLYSINS    AND    ILEMOLYSINS 

Historical. — As  far  back  as  1874,  Gscheidlen 
and  Traube  l  demonstrated  that  considerable  quan- 
tities of  septic  material  could  be  injected  into  the 
circulation  of  warm-blooded  animals  without 
apparently  any  effect  on  the  animal.  Very  little 
was  thought  of  this  observation  at  the  time,  and  it 
is  not  until  more  than  ten  years  later  that  we  find 
a  similar  observation  made  by  Fodor.2  In  1888 
Nuttall 3  showed  that  normal  blood  serum  possessed 
marked  germicidal  properties,  and  his  observations 
stimulated  a  number  of  workers  who  undertook  to 
determine  the  conditions  most  favorable  to  the 
exhibition  of  this  phenomenon,  and  further  to 
decide  upon  the  constituent  of  the  serum  to  which 
this  property  was  due  or  whether  it  was  a  function 
of  the  serum  as  a  whole.  In  1889  Buchner  4  pub- 
lished a  series  of  experiments  and  showed  that  an 
exposure  of  55°  C.  robs  the  serum  of  its  bacteri- 
cidal property.  He  concluded  that  the  active 
element  in  the  process  was  a  living  albumen  and 

1  Gscheidlen    and    Traube.     Schlez.    Gesellschaft.    f.    Vater- 
land.  Cultur,  Med.  Sect.,  1874. 

2  Fodor,  Deutsche  med.  Wochenschr,  1886. 

3  Nuttall,  Zeitschr.  f.  Hygiene,  Vol.  iv,  1888. 

4  Buchner,  Centralblatt  Bacteriologie,  Vol.  v,  1889.     Archiv. 
f.  Hygiene,  Vol.  x,  1890. 

5° 


BACTERIOLYSINS  AND   HALMOLYSINS  51 

suggested  for  it  the  name  "  alexin."  He  found  that 
it  was  possible  to  greatly  increase  the  bactericidal 
action,  (i.e.  the  quantity  of  "  alexin  ")  for  a  par- 
ticular bacterium  by  immunizing  an  animal  with 
that  bacterium. 

Pfeiff er's  Phenomenon.  —  An  enormous  advance 
in  the  study  of  immunity  was  made  in  the  dis- 
covery of  Pfeiffer's  phenomenon  in  1894,  and  it  is 
to  Pfeiffer's  splendid  observations  1  that  we  owe 
the  first  and  most  important  insight  into  the  mode 
of  action  .  of  the  bacteriolytic  immune  sera.  A 
normal  guinea  pig  is  able  to  kill  and  dissolve  a 
number  of  living  cholera  spirilla  if  these  are  in- 
jected intraperitoneally.  If  in  such  an  animal  we 
gradually  increase  the  dose  injected,  it  will  be  pos- 
sible after  a  time  to  inject  at  one  dose  an  amount 
of  cholera  spirilla  that  represents  many  times  an 
ordinary  fatal  dose.  If  from  this  animal  we  now 
withdraw  serum  and  inject  it  into  another  animal, 
we  find  that  this  serum,  even  in  such  small  amounts 
as  the  fractional  part  of  a  centigram  or  even  of  a 
milligram,  is  able  to  protect  the  second  animal 
against  living  cholera  spirilla.  Under  the  influence 
of  these  small  amounts  of  serum  of  the  treated  ani- 
mal, the  organism  of  the  untreated  animal  is  able 
to  dissolve  large  amounts  of  cholera  spirilla,  amounts 
which  would  otherwise  be  invariably  fatal.  This 
process,  as  R.  Pfeiff er  showed,  is  a  specific  one,  i.e., 

1  R.  Pfeiffer,  Zeitschr.     Hygiene,  Vol.  xviii,  1894. 


5 2  IMMUNE   SERA 

the  serum  of  the  guinea  pig  treated  with  cholera 
spirilla  transmits  an  increased  solvent  power  only 
for  cholera  spirilla,  but  not  for  any  other  species  of 
bacteria.  The  active  substance  of  such  a  bacterio- 
lytic  immune  serum  Pfeiffer  called  a  specific  bac- 
tericide.  If  we  allow  some  of  this  specific  cholera 
immune -serum  to  remain  for  some  time  outside  of 
the  body,  e.g.  in  a  bottle,  and  then  test  it  for 
solvent  properties  against  cholera  spirilla,  not  in  a 
living  body  but  in  a  test-tube,  we  shall  find  that  its 
power  is  almost  nil.  If  we  add  to  this  serum  in 
the  test-tube  some  fresh  peritoneal  exudate  or 
some  other  body  fluid,  suoh  as  serum  of  a  normal, 
untreated  guinea  pig,  as  Metchnikoff  first  did,  we 
find  that  this  serum  has  now  acquired  the  power 
to  rapidly  dissolve  cholera  spirilla  even  in  a  test- 
tube.  Bordet,1  in  1895,  showed  that  in  order  for 
the  specific  immune  serum  to  dissolve  spirilla  in  a 
test-tube,  it  is  unnecessary  to  add  fresh  normal 
serum  or  peritoneal  fluid;  but  that  immune  serum 
freshly  drawn  from  the  vein  is  able  even  under 
these  circumstances  to  dissolve  the  spirilla. 

Haemolysis.  —  In  his  experiments  with  the  bac- 
teriolysis of  cholora  spirilla,  Bordet  used  as  an 
immune  serum  the  serum  of  a  goat  that  had  been 
immunized  against  cholera  spirilla,  and  as  alexin, 
fresh  normal  guinea-pig  serum.  It  often  happened 
that  the  latter  contained  a  certain  number  of  red 

1  Bordet,  Annal.  Inst.  Pasteur,  1895. 


BACTERIOLYSINS  AND  HsEMOLYSINS  53 

blood  cells  and  he  found  that  these  were  agglutinated 
and  would  be  agglutinated  even  when  mixed  with 
normal  goat  serum.  Knowing,  as  he  did,  that 
immunization  against  bacteria  increases  the  agglu- 
tinating property  toward  a  given  organism  over 
that  in  the  normal  animal,  it  was  natural  that  he 
should  experiment  to  see  whether  similar  results 
could  be  obtained  with  red  blood  cells.  Accord- 
ingly he  injected  guinea  pigs  several  times  with 
5  cc.  defibrinated  rabbit's  blood,  and  found  that 
not  only  did  this  guinea-pig  serum  acquire  agglu- 
tinating properties,  but  also  the  property  to  dissolve 
rapidly  and  intensely,  in  a  test- tube,  the  red  blood 
cells  of  a  rabbit.  The  serum  of  a  normal  guinea 
pig  was  incapable  of  doing  this,  or  did  it  in  only  a 
slight  degree.  Bordet  could  further  show  that  this 
action  is  a  specific  one,  i.e.,  the  serum  of  animals 
treated  with  rabbit  blood  acquires  this  dissolving 
property  only  for  the  red  cells  of  rabbits,  not  for 
those  of  any  other  species  of  animal.  '  For  the 
latter,  such  a  serum  is  no  more  strongly  solvent 
than  the  serum  of  a  normal  animal.  The  same 
property  that  Bordet  had  demonstrated  in  the  serum 
of  guinea  pigs  treated  with  rabbit  blood  could  now 
be  shown  for  the  sera  of  all  animals  treated  with 
blood  cells  of  a  different  species.  We  can  formulate 
this  as  follows:  The  serum  of  animals,  species  A, 
after  these  have  been  injected  either  subcutaneously, 
intraperitoneally,  or  intravenously  with  erythrocytes 


54 


IMMUNE  SERA 


of  species  B,  acquires  an  increased  solvent  action 
for  erythrocytes  of  species  B,  and  only  for  this 
species.1  It  is  therefore  a  specific  action.  We  call 
this  hcemolysis,  and  the  substances  which  effect 
the  solution  of  the  red  cells,  hcemolysins  or  hczmo- 
toxins. 

At  about  the  same  time,  and  independently  of 
Bordet,  similar  experiments  with  similar  results 
were  published  by  Landsteiner  2  and  v.  Dungern.3 
As  a  result  of  this  work,  the  acquired  toxicity  of 
horse  serum,  found  by  Belfanti  and  Carbone  when 
they  treated  horses  with  red  cells  of  rabbits,  was 
explained.  The  serum  of  the  horses  so  treated  had 
become  h&molytic  for  rabbit  blood,  and  therefore 
caused  a  solution  or  destruction  of  the  red  cells 
in  the  living  body  just  as  it  did  in  a  test-tube. 

Nature  of  Hcemolytic  Sera.  —  In  a  subsequent 
study  Bordet 4  was  able  to  show  that  the  sol- 
vent power  of  the  specific  haemolysins  depended 
on  the  combined  action  of  two  constituents  of  the 
specific  serum.  When  the  fresh  haemolytic  serum 
was  heated  for  half  an  hour  to  55°  C.,  it  lost  its 
power.  If  to  this  inactive  serum  a  very  small 
amount  of  the  serum  of  a  normal  guinea  pig  was 
added  (a  serum  which  of  course  was  not  haemolytic 
for  rabbit  red  cells),  the  full  haemolytic  power  was 

1  We  shall  point  out  a  few  exceptions  later  on. 

2  Landsteiner,  Centralblatt  Bacteriol.,  Vol.  xxv,  1899. 

3  Von  Dungern,  Munch,  med.  Wochenschrift,  1898. 

4  Bordet,  Annal.  Inst.  Pasteur,  Vol.  xii,  1898. 


BACTERIOLYSIS S   AND  H&MOLYSINS          55 

restored  to  this  inactive  serum.  In  other  words, 
it  had  been  reactivated  by  this  addition. 

This  experiment  permits  of  only  one  conclusion, 
namely,  that  the  hDemolytic  action  of  the  specific 
haemolytic  serum  depends  on  two  substances.  One 
of  these  is  able  to  withstand  heating  to  55°C.,  and 
is  contained  only  in  the  specific  serum.  The  other 
is  destroyed  by  heating  to  55°  C.,  and  is  contained 
not  only  in  the  specific  haemolytic  serum,  but  also 
in  the  serum  of  normal  untreated  animals. 

Buchner,  we  have  seen,  applied  the  term  alexins 
to  the  constituents  of  normal  serum  which  were 
actively  destructive  to  corpuscular  elements,  bac- 
teria, and  other  cells  with  which  they  came  in  con- 
tact. This  term  was  retained  by  Bordet  to  desig- 
nate that  constituent  of  normal  serum  which  did 
not  withstand  heating  to  55°  C.,  and  which  was  one 
of  the  factors  in  the  haemolytic  process.  The  other 
substance,  which  was  found  only  in  the  specific 
serum  and  which  withstood  heating  to  55°C.,  he 
termed  substance  sensibilatrice. 

According  to  Bordet,  therefore,  the  substances 
required  for  haemolysis  are  the  substance  sensibila- 
trice  of  the  specific  haemolytic  serurn  and  the 
alexin  which  exists  even  in  normal  serum.  The 
action  of  these  two  substances  Bordet  explains  by 
assuming  that  the  red  cell  is  not  vulnerable  to  the 
alexin ;  just  as,  for  example,  there  are  certain  sub- 
stances that  will  not  take  a  dye  without  the  previous 


56  IMMUNE  SERA 

action  of  a  mordant.  The  substance  sensibilatrice 
plays  the  role  of  mordant.  It  makes  the  blood 
cells  vulnerable  to  the  alexin,  so  that  the  latter  can 
attack  the  cells  and  dissolve  them.  The  alexin  he 
regards  as  a  sort  of  ferment  body  with  digestive 
powers. 

Bordet  says  further,  that  the  substance  sensi- 
bilatrice sensitizes  the  blood  cells  not  only  for  the 
alexin  derived  from  the  serum  of  the  same  species 
as  that  from  which  it  (the  substance  sensibilatrice) 
is  derived,  but  sensitizes  such  cells  also  for  the 
alexins  of  normal  sera  of  other  species.  For  ex- 
ample, in  the  foregoing  experiment  of  Bordet,  the 
substance  sensibilatrice  derived  from  the  guinea 
pig  by  treatment  with  rabbit  blood  sensitizes  the 
red  blood  cells  of  rabbits  not  only  for  the  alexin 
of  normal  guinea  pig  blood,  but  also  for  the  alsxins 
of  other  normal  sera.  In  another  experiment 
this  author  showed  that  rabbit  red  cells  sensitized 
with  an  inactive  specific  haemolytic  serum  derived 
from  a  guinea  pig  would  dissolve  rapidly  on  the 
addition  of  normal  rabbit  blood.  Here,  then,  the 
rabbit  red  cells,  sensitized  (according  to  Bordet)  by 
the  substance  sensibilatrice  of  the  guinea  pig,  dissolve 
on  the  addition  of  the  alexin  of  their  own  serum. 

Resume. — Reviewing  the  important  facts  we  have 
learned,  we  find  them  to  be  as  follows:  By  means 
of  the  treatment  of  one  species  of  animal  with  the 
erythrocytes  of  a  different  one,  the  serum  of  the 


B ACT ERIOLY SINS  AND   H&MOLYSINS  $7 

first  species  acquires  an  uncommonly  increased 
power  to  dissolve  and  to  agglutinate  the  erythro- 
cytes  of  the  second  species.  This  increased  hsemo- 
lytic  power  shows  itself  not  only  in  vivo,  so  that  an 
animal  so  treated  is  able  to  cause  red  cells  injected 
into  it  to  rapidly  dissolve  and  disappear,  but  it 
shows  itself  also  in  vitro  when  the  serum  of  this 
animal  is  used.  The  process  consists  in  the  com- 
bined action  of  two  substances,  that  which  is  excited 
in  response  to  the  injection,  the  substance  sensi- 
bilatrice,  and  the  alexin  of  normal  serum. 

Analogy  between  the  Bacteriolytic  and  Haemolytic 
Processes. —  If  we  now  recall  the  main  points  in 
cholera  immunity  the  close  analogy  between  this  and 
the  subject  of  haemolysis  is  apparent.  Just  as,  when 
immunizing  an  organism  against  cholera  bacilli 
the  organism  responds  with  an  increased  solvent 
power  for  those  bacteria,  so  does,  the  organism 
respond  when  it  is  treated,  i.e.  immunized,  with 
red  cells  of  another  species,  by  increasing  the  sol- 
vent power  of  its  serum  for  those  particular  cells. 
Furthermore,  just  as  the  haemolytic  process  was 
seen  to  depend  on  the  combined  action  of  two  sub- 
stances, one  developed  in  the  hagmolytic  serum, 
the  other  already  present  in  normal  serum,  so  also 
in  the  bactericidal  process  just  studied  there  are 
two  factors.  It  is  easy  to  understand,  therefore, 
what  formerly  was  not  at  all  clear,  why  a  specific 
bactericidal  serum  against  cholera,  typhoid,  or 


58  IMMUNE  SERA 

other  infectious  disease  should  not  act  in  a  test- 
tube  unless  there  had  first  been  added  some  normal 
serum  (according  to  Metchnikoff),  or  there  had 
been  employed  a  perfectly  fresh  serum  (according 
to  Bordet) :  simply  because  in  either  of  these 
ways  the  alexin  necessary  to  co-operate  with  the 
substance  sensibilatrice  is  introduced.  This  alexin 
no  longer  exists  in  the  immune  serum,  if  this  be 
not  perfectly  fresh,  for  we  have  seen  that  it  decom- 
poses either  on  warming,  or  spontaneously  on  stand- 
ing. A  bactericidal  serum,  therefore,  that  has 
stood  for  some  time  is  incapable  of  dissolving 
bacteria.  It  is  possible,  however,  to  make  an  old 
inactive  serum  again  capable  of  dissolving  bacteria 
in  vitro  by  adding  a  little  fresh  alexin,  according 
to  the  suggestion  of  Metchnikoff.  In  other  words, 
it  is  thus  reactivated.  Another  obscure  point  was 
cleared  up  by  these  studies:  why  a  specific  bac- 
tericidal serum  which  is  inactive  in  vitro  should 
be  intensely  active  in  the  living  body.  This  is 
because  in  the  living  body  the  serum  finds  the  alexin 
necessary  for  its  working,  which  is  not  the  case  in 
the  test-tube  unless  fresh  normal  serum  be  added. 
We  see  from  all  this  that  even  the  first  experiments 
in  haemolysis  have  served  to  clear  up  a  number  of 
practical  points  in  an  important  branch  of  bacteri- 
ology. 

Ehrlich  and  Morgenroth  on  the  Nature  of  Haemo- 
lysis. —  In  continuing  the  study  of  hsemolysins  we 


BACTERIOLYSINS  AND  H.EMOLYS1NS          59 

must  note  particularly  the  researches  of  Ehrlich 
and  Morgenroth.1  These  authors  asked  themselves 
the  following  questions:  (i)  What  relation  does 
the  haemolytic  serum  or  its  two  active  components 
bear  to  the  cell  to  be  dissolved?  (2)  On  what 
does  the  specificity  of  this  haemolytic  process 
depend  ?  Ehrlich  was  led  to  these  researches  partic- 
ularly by  his  so-called  Side-chain  Theory,  which 
we  shall  examine  in  a  moment. 

He  made  his  experiments  with  a  hasmolytic 
serum  that  had  been  derived  from  a  goat  treated 
with  the  red  cells  of  a  sheep.  This  serum,  there- 
fore, was  haemolytic  specifically  for  sheep  blood 
cells;  i.e.,  it  had  increased  solvent  properties  exclu- 
sively for  sheep  blood  cells. 

Basing  his  reasoning  on  his  side-chain  theory, 
Ehrlich  argued  as  follows:  "  If  the  haemolysin  is 
able  to  exert  a  specific  solvent  action  on  sheep 
blood  cells,  then  either  of  its  two  factors,  the  sub- 
stance sensibilatrice  of  Bordet  or  the  alexin  of  nor- 
mal serum,  must  possess  a  specific  affinity  for  these 
red  cells.  It  must  be  possible  to  show  this  experi- 
mentally." Such  in  fact  is  the  case,  and  the  experi- 
ments devised  by  him  are  as  follows : 

Experiment  i .  —  Ehrlich  and  Morgenroth,  as 
already  said,  experimented  with  a  serum  that  was 
specifically  haemolytic  for  sheep  blood  cells.  They 

1  See  the  various  papers  in  "Collected  Studies  on  Immunity," 
Ehrlich- Bolduan,  Wiley  &  Sons,  New  York,  1910. 


60  IMMUNE   SERA 

made  this  inactive  by  heating  to  55°  C.,  so  that  then 
it  contained  only  the  substance  sensibilatrice. 
Next  they  added  a  sufficient  quantity  of  sheep 
red  cells,  and  after  a  time  centrifuged  the  mixture. 
They  were  now  able  to  show  that  the  red  cells  had 
combined  with  all  the  substance  sensibilatrice,  and 
that  the  supernatant  clear  liquid  was  free  from  the 
same.  In  order  to  prove  that  such  was  the  case 
they  proceeded  thus:  To  some  of  the  clear  centri- 
fuged fluid  they  added  more  sheep  red  cells;  and, 
in  order  to  reactivate  the  serum,  a  sufficient  amount 
of  alexin  in  the  form  of  normal  serum  was  also 
added.  The  red  cells,  however,  did  not  dissolve  — 
there  was  no  substance  sensibilatrice.  The  next 
point  to  prove  was  that  this  substance  had  actually 
combined  with  the  red  cells.  The  red  cells  which 
had  been  separated  by  the  centrifuge  were  mixed 
with  a  little  normal  salt  solution  after  freeing  them 
as  much  as  possible  from  fluid.  Then  a  little  alexin 
in  the  form  of  normal  serum  was  added.  After 
remaining  thus  for  two  hours  at  3  7°  C.  these  cells 
had  all  dissolved. 

In  this  experiment,  therefore,  the  red  cells  had 
combined  with  all  the  substance  sensibilatrice, 
entirely  freeing  the  serum  of  the  same.  That  the 
action  was  a  chemical  one  and  not  a  mere  absorp- 
tion was  shown  by  the  fact  that  red  blood  cells  of 
other  animals,  rabbits  or  goats  for  example,  exerted 
no  combining  power  at  all  when  used  instead  of 


BACTERIOLYSINS   AND   H&MOLYSINS  6l 

the  sheep  cells  in  the  above  experiment.  The 
union  of  these  cells,  morever,  is  such  a  firm  one 
that  repeated  washing  of  the  cells  with  normal  salt 
solution  does  not  break  it  up. 

So  far  as  concerns  the  technique  of  the  experi- 
ments, I  should  like  to  observe  that  the  addition 
of  red  cells  in  this  as  well  as  in  all  the  following 
experiments  was  always  in  the  form  of  a  5%  mix- 
ture or  suspension  in  0.85%,  i.e.,  isotonic,  salt  solu- 
tion. 

The  second  important  question  solved  by  these 
authors  was  this;  What  relation  does  the  alexin 
bear  to  the  red  cells  ?  They  studied  this  by  means 
of  a  series  of  experiments  similar  to  the  preceding. 

Experiment  2.  —  Sheep  blood  was  mixed  with 
normal,  i.e.  not  haemolytic,  goat  serum.  After  a 
time  the  mixture  was  centrifuged  and  the  two  por- 
tions tested  with  substance  sensibilatrice  to  deter- 
mine the  presence  of  alexin.  It  was  found  that  in 
this  case  the  red  cells  acted  quite  differently.  In 
direct  contrast  to  their  behavior  toward  the  sub- 
stance sensibilatrice  in  the  first  experiment,  they 
now  did  not  combine  with  even  the  smallest  por- 
tion of  alexin,  and  remained  absolutely  unchanged. 

Experiment  3.  --The  third  series  of  experiments 
was  undertaken  to  show  what  relations  existed 
between  the  blood  cells  on  the  one  hand,  and  the 
substance  sensibilatrice  and  the  alexin  on  the 
other,  when  both  were  present  at  the  same  time, 


62  IMMUNE  SERA 

and  not,  as  in  the  other  experiments,  when  they 
were  present  separately.  This  investigation  was 
complicated  by  the  fact  that  the  specific  immune 
serum  very  rapidly  dissolves  the  red  cells  for  which 
it  is  specific,  and  that  any  prolonged  contact  be- 
tween the  cells  and  the  serum,  in  order  to  effect 
binding  of  the  substance  sensibilatrice,  is  out  of 
the  question.  Ehrlich  and  Morgenroth  found  that 
at  o°  C.  no  solution  of  the  red  cells  by  the  haemo- 
lytic  serum  takes  place.  They  therefore  mixed  some 
of  their  specific  hsemolytic  serum  with  sheep  blood 
cells,  and  kept  this  mixture  at  o°-3°  C.  for  sev- 
eral hours.  No  solution  took  place.  They  now 
centrifuged  and  tested  both  the  sedimented  red 
cells  and  the  clear  supernatant  serum.  It  was 
found  that  at  the  temperature  o°-3°  C.  the  red 
cells  had  combined  with  all  of  the  substance  sen- 
sibilatrice, but  had  left  the  alexin  practically 
untouched. 

It  still  remained  to  show  the  relation  of  these 
two  substances  to  the  red  cells  at  higher  temper- 
atures. At  37°-4o°  C.,  as  already  mentioned, 
haemolysis  occurs  rapidly,  beginning  usually  within 
fifteen  minutes.  It  was  possible,  therefore,  to 
leave  the  cells  and  serum  in  contact  for  not  over 
ten  minutes.  Then  the  mixture  was  centrifuged 
-as  before.  The  sedimented  blood  cells  mixed  with 
normal  salt  solution  showed  haemolysis  of  a  moder- 
ate degree.  The  solution  became  complete  when 


'BACTERtOLYSINS  AND  HMMOLYSMS         63 

a  little  normal  serum  was  added.  The  supernatant 
clear  fluid  separated  by  the  centrifuge  did  not  dis- 
solve sheep  red  cells.  On  the  addition,  however, 
of  substance  sensibilatrice  it  dissolved  them  com- 
pletely. 

From  this  experiment  Ehrlich  concludes  that  the 
substance  sensibilatrice  possesses  one  combining 
group  with  an  intense  affinity  (active  even  at  o°  C.), 
for  the  red  cell,  and  a  second  group  possessing  a 
weaker  affinity  (one  requiring  a  higher  temperature) 
for  the  alexin. 

Nomenclature. — In  place  of  the  name  substance 
sensibilatrice  Ehrlich  first  introduced  the  term 
immune  body;  later  on  he  called  it  the  amboceptor, 
to  express  the  idea  that  it  served  as  a  link  between 
alexin  and  cell.  Other  names  proposed  for  this  sub- 
stance have  been  substance  fixatrice  by  Metchnikoff, 
copula,  desmon,  preparator  by  Muller.  Instead  of 
the  name  alexin,  Ehrlich  now  uses  the  term  com- 
plement in  order  to  express  the  idea  that  this  body 
completes  the  action  of  the  immune  body. 

According  to  Ehrlich  the  red  blood  cells  possess 
specific  affinity  for  the  immune  body,  but  none 
whatever  for  the  alexin.  The  alexin,  therefore, 
possesses  no  combining  group  which  can  attach  itself 
directly  to  the  red  blood  cell.  It  acts  on  these  cells 
only  through  an  intermediary,  the  immune  body, 
which  therefore  must  possess  two  binding  groups  one 
of  which  attaches  to  the  red  blood  cell  and  the  other  to 


64  IMMUNE   SERA 

the  alexin  of  normal  serum.  As  already  stated,  the 
group  which  attaches  to  the  red  blood  cell  possesses 
a  much  stronger  affinity  than  that  which  combines 
with  the  alexin.  This  follows  from  the  last  two 
experiments  of  Ehrlich  before  cited,  in  which  he 
showed  that  at  the  lower  temperature,  and  with 
both  substances  present  with  the  blood  cells,  only 
the  immune  body  combined  with  the  cells,  while 
the  alexin  remained  uncombined.  At  the  higher 
temperature  the  alexin  also  exerted  its  affinity,  foi 
then  the  red  cells  combined  with  all  the  immune 
body  and  with  part  of  the  alexin.  We  saw  that 
after  a  time  the  red  cells  partially  dissolved,  but 
that  complete  solution  occurred  only  after  some 
fresh  alexin  had  been  added.  This  showed  that 
although  the  red  cells  had  combined  with  all  the 
immune  body  necessary  for  their  solution,  they  had 
been  unable  to  bind  all  the  alexin  necessary.  We 
may  say,  therefore,  that  that  group  of  the  immune 
body  wrhich  combines  with  the  red  cell  has  a 
stronger  affinity  than  that  which  combines  with  the 
alexin. 

Role  of  the  Immune  Body.  —  According  to  Ehrlich, 
then,  the  role  of  the  immune  body  consists  in  this, 
that  it  attaches  itself  to  the  red  cell  on  the  one  hand, 
and  to  the  complement  on  the  other,  and  in  this  way 
brings  the  digestive  powers  of  the  latter  to  bear 
upon  the  cell,  the  complement  possessing  no  affinity 
for  the  red  cell.  Immune  body  and  complement 


BACTERIOLYSINS  AND   H^EMOLYSINS         65 

have  no  very  great  affinity  for  each  other.  At  o°  C. 
they  may  exist  in  serum  side  by  side,  and  they 
combine  only  at  higher  temperatures. 

The  amount  of  immune  body  which  combines 
with  the  red  cells  may  vary  greatly,  as  the  experi- 
ments of  Bordet  and  of  Ehrlich  clearly  show. 
Some  red  cells  combine  with  only  just  enough 
immune  body  to  effect  their  solution.  Others  are 
able  to  so  saturate  themselves  with  immune  body 
that  they  may  have  a  hundred  times  the  amount 
necessary  for  their  solution. 

On  what  the  Specificity  Depends.  —  From  the  pre- 
ceding it  follows  that  the  specific  action  of  the 
haemolytic  sera,  and,  I  may  at  once  add,  of  the  bac- 
tericidal sera  also,  is  due  exclusively  to  the  immune 
body.  This  possesses  a  combining  group  which  is 
specific  for  the  cells  with  which  the  animal  was 
treated;  e.g.,  the  combining  group  of  an  immune 
body  produced  by  treatment  with  rabbit  blood 
will  fit  only  to  a  certain  group  in  the  blood  cells  of* 
rabbits;  an  immune  body  produced  by  treatment 
with  chicken  blood  will  fit  only  to  parts  of  the  red 
cells  of  chickens;  one  produced  by  treating  an  ani- 
mal with  cholera  bacilli  will  fit  only  to  this  species 
of  bacteria  and  combine  only  with  the  members  of 
it.  Keeping  to  the  well-known  simile  of  Emil 
Fischer,  the  relation  is  like  that  between  lock  and 
key,  each  lock  being  fitted  only  by  a  particular 
key. 


66  IMMUNE   SERA 

To  repeat  —  for  the  point  is  of  the  greatest 
importance  —  the  role  of  the  immune  body  consists 
in  tying  the  complements  of  normal  serum,  which 
have  no  affinity  for  the  red  cells  or  for  the  bacteria, 
indirectly  to  these  cells  so  that  their  solution  and 
digestion  may  be  effected  by  the  complements. 
In  other  words,  the  immune  body  serves  to  con- 
centrate on  the  corpuscular  element  to  be  dis- 
solved all  the  widely  distributed  complement  found 
in  normal  serum. 

Ehrlich's  conception  of  the  relation  existing  be- 
tween complement,  immune  body  (i.e.,  amboceptor) 
and  erythrocyte  is  shown  in  the  accompanying 
figure. 

i.  H. 

symotoxic  group 


\ 


COMPLEMENT—      ^ 

— haptophore  group 
— complementophile  gr. 
IMMUNE  BODY—     ~ 

:ytophile  group 
• — receptor 


CEL1 

FIG.  7 

Difference  between  a  Specific  Serum  and  a  Normal 
One.  —  The  difference,  then,  between  a  specific 
haemolytic  or  a  specific  bactericidal  serum  and  a 
normal  one  consists  in  this  — *  that  the  specific  serum 
contains  an  immune  body  which  is  specific  for  a 


BACTERIOLYSINS  AND  H&MOLYS1NS          67 

certain  cellular  clement  and  by  means  of  which  the 
complement  present  in  all  normal  serum  can  be  con- 
centrated on  this  element  to  cause  its  solution.  We 
shall  return  to  this  subject  later. 

Diverging  Views  of  Ehrlich  and  Bordet.  —  Now  if 
we  recall  the  first  experiments  of  Bordet  and  his 
conclusions  respecting  the  manner  in  which  the 
factors  concerned  a^.ted,  we  shall  at  once  see  how 
Ehrlich  and  Bordet  differ.  Bordet  assumes  that 
the  substance  sensibilatrice  (the  immune  body) 
acts  as  a  kind  of  mordant  on  the  red  cells  or  bac- 
teria, sensitizing  these  to  the  action  of  the  alexin 
(complement).  That  is  to  say,  neither  the  cell  nor 
the  immune  body  has  alone  any  manifest  affinity 
for  the  alexin,  but  they  form  by  their  union  a 
complex  which  can  absorb  alexin,  in  other  words 
which  has  particular  properties  of  adhesion.  Accord- 
ing to  Bordet,  then,  there  is  no  such  thing  as  an 
amboceptor,  and  no  complementophile  group.  He 
cites  the  experiments  of  Muir  as  showing  that  the 
hypothesis  of  a  complementophile  group  is  untenable. 
This  author  found  that  blood  corpuscles  which  had 
fixed  the  sensitizer  (immune  body)  and  had  been 
saturated  with  alexin  could  subsequently,  by  dif- 
fusion, lose  a  certain  amount  of  their  sensitizer, 
although  they  retain  the  complement,  and  what  is 
more,  in  this  instance  they  lose  as  much  sensitizer 
as  if  they  had  not  absorbed  complement.  Con- 
sequently, says.  Bordet,  it  is  in  no  way  through  the 


68  IMMUNE   SERA 

mediation  of  the  sensitizer  that  the  alexin  attaches 
itself  to  the  corpuscles;  if  this  were  the  case  the 
removal  of  the  sensitizer  would  necessarily  imply 
that  of  the  alexin  (complement) ,  which,  as  we  have 
just  mentioned,  does  not  leave  the  corpuscles. 

According  to  Ehrlich,  however,  the  process  is  not 
analogous  to  a  staining  process,  but  follows  definite 
laws  of  chemical  combination,  there  being,  in  fact, 
no  affinity  whatever  between  the  complement  and 
the  blood  cells  or  bacteria.  Furthermore,  according 
to  this  authority,  the  complement  always  acts 
through  the  mediation  of  the  immune  body,  which 
possesses  two  combining  groups;  one,  the  cytophile 
group,  combining  with  the  cell,  and  another,  the 
complementophile  group,  combining  with  the  com- 
plement. 

The  Side-Chain  Theory  Applied  to  these  Bodies.  - 
All  of  the  specific  relations  which,  in  a  previous 
chapter,  we  saw  existed  between  toxin  and  anti- 
toxin, Ehrlich  and  Morgenroth  in  their  experi- 
ments above  noted  found  existed  also  between 
immune  body  and  the  specific  blood  cell.  The 
immune  body  must  therefore  possess  a  haptophore 
group  which  fits  exactly  to  certain  receptors  or 
side  chains  of  the  red  cells,  just  as  the  anti-body 
according  to  the  side-chain  theory  possesses  a 
group  that  fits  exactly  into  the  specific  combining 
group  —  i.e.,  haptophore  group  —  of  the  toxin  or 
toxoid  used  for  exciting  the  immunity. 


BACTERIOLYSINS   AND    H&MOLYS1NS         69 

If,  for  example,  we  produce  a  haemolytic  serum 
specific  for  red  cells  of  a  rabbit  by  injecting  an 
animal  with  these  cells,  the  haptophore  groups  of 
this  serum,  i.e.,  the  free  side  chains  thrust  off,  must 
possess  specific  combining  relations  with  the  red 
cells  of  rabbits.  That  such  is  the  case  in  the  haemo- 
lytic immune  serum  we  saw  from  the  experiments 
of  Ehrlich  and  Morgenroth. 

In  consequence  of  all  this,  Ehrlich  widened 
the  application  of  his  side-chain  theory  so  as  to 
include  not  only  the  production  of  antitoxin  but 
also  the  production  of  bactericidal,  haemolytic, 
and  other  immune  bodies.  He  expressed  this 
somewhat  as  follows:  //  any  substance,  be  it  toxin, 
ferment,  constituent  of  a  bacterial  or  animal  cell,  or 
of  animal  fluid,  possess  the  power  by  means  of  a 
fitting  haptophore  group  to  combine  with  side  chains 
(receptors)  of  the  living  organism,  the  possibility  for 
the  overproduction  and  throwing  off  of  these  recep- 
tors is  given,  i.e.,  the  possibility  to  produce  a  cor- 
responding anti-body. 

Specific  anti-bodies  in  the  serum  as  a  result  of 
immunizing  processes  can  only  be  produced,  there- 
fore, by  substances  which  possess  a  haptophore 
group  l  and  which,  in  consequence,  are  able  to  form  a 
firm  union  with  a  definite  part  of  the  living  or- 
ganisms, the  receptor.  This  is  not  the  case  with 

1  Such  substances  Ehrlich  terms  ''haptins."  See  page 
16. 


70  IMMUNE  SERA 

alkaloids,  e.g.,  morphine,  strychnine,  etc.,  which 
according  to  Ehrlich  enter  into  a  loose  union,  a  kind 
of  solid  solution  with  the  cells.  It  is  for  this  reason 
that  we  are  unable  to  produce  any  anti-bodies  in 
the  blood  serum  against  these  poisons.  Ehrlich 
says  further  that  all  of  the  substances  taking  part 
in  the  production  of  immunity,  including  of  course 
complement  and  immune  body,  have  certain 
definite  affinities  for  each  other,  and  in  order 
to  act  they  must  fit  stereochemically  to  each 
other. 

As  we  have  already  seen,  we  are  able  by  means 
of  the  injection  of  a  variety  of  substances  or  cells 
to  produce  a  similar  variety  of  immune  bodies  in 
the  serum.  Thus  we  can  immunize  a  rabbit  so 
that  its  serum  will  possess  specific  haemolytic 
bodies  against  the  red  cells  of  guinea  pigs,  goats, 
chickens,  and  oxen  and  specific  bactericidal 
bodies  against  cholera  and  typhoid  bacilli,  etc., 
and  as  we  shall  see,  still  other  groups  of  anti- 
bodies. 

Multiplicity  of  Complements.  —  Under  these  cir- 
cumstances an  important  question  presents  itself: 
Is  there  in  normal  serum  one  single  complement 
which  completes  the  action  of  all  these  various 
immune  bodies,  one,  for  example,  which  in  the 
above  illustration  will  fit  all  the  haemolytic  immune 
bodies  as  well  as  all  the  bactericidal  ones,  or 
are  there  a  great  many  different  complements? 


BACTERIOLYSINS  AND  HMMOLYSINS          71 

Ehrlich,  as  a  result  of  his  experimental  work 
with  Morgenroth,  claims  that  the  latter  is  the  case ; 
namely,  that  it  takes  a  different  complement  to  fit 
the  immune  body  specifically  haemolytic  for  guinea 
pig  blood  than  it  does  to  fit  that  specific  for  chicken 
blood. 

Bordet,  on  the  other  hand,  assuming  that  the 
immune  body  plays  the  role  of  mordant,  believes 
that  there  is  but  one  single  complement  in  the 
serum.  According  to  him,  this  complement  is 
able  to  dissolve  blood  cells  as  well  as  bacteria  after 
these  have  been  sensitized  by  their  specific  immune 
body.  Each  of  these  authors  supports  his  claims 
by  means  of  ingenious  experiments,  for  the  details 
of  which,  however,  we  must  refer  to  the  original 
articles,  as  they  require  the  knowledge  of  a  specialist 
for  their  comprehension.  We  shall,  however,  give 
one  of  Bordet 's  l  experiments  on  this  point  in  some 
detail,  since  it  has  found  extensive  application  in 
another  direction. 

The  Bordet-Gengou  Phenomenon.  — Bordet  sensi- 
tized blood  corpuscles  with  appropriate  amboceptors, 
and  then  exposed  them  to  the  action  of  a  freshly  drawn 
normal  serum.  If  now  he  waited  for  the  occurrence 
of  haemolysis  and  then  added  sensitized  cells  (bacteria 
or  blood  corpuscles  of  a  different  species),  the  latter  re- 
mained entirely  unchanged,  although  the  serum  that  had 
been  used  as  complement  was  capable  in  its  original  con- 

1  Bordet  and  Gengou,  Annal.  Inst.  Pasteur.     Vol.  xv,  1901. 


72  IMMUNE  SERA 

dition  of  destroying  these  also.  When  fresh  serum  was 
first  brought  into  contact  with  sensitized  bacteria,  simi- 
lar results  were  obtained.  The  blood  corpuscles  sub- 
sequently added  did  not  then  undergo  haemolysis. 
//  such  an  action  on  one  of  the  sensitive  substrata  has 
once  taken  place,  the  active  sera,  as  a  rule,  are  deprived 
of  all  their  complement  functions,  from  which  Bordet 
concludes  that  the  destruction  of  the  most  varied 
elements  by  one  and  the  same  serum  must  be  due  to  a 
single  complement. 

It  may  be  said  in  passing  that  Ehrlich  admits  the 
correctness  of  the  above  experimental  results,  but 
brings  forward  arguments  to  show  that  Bordet 's  inter- 
pretation as  to  the  existence  of  only  a  single  complement 
cannot  be  accepted. 

This  experiment  of  Bordet  is  usually  spoken  of  as 
the  "  Bordet-Gengou  phenomenon  "  and  is  now  used 
largely  in  determining  whether  or  not  a  given  serum 
possesses  certain  amboceptors.  The  serum  to  be 
tested  is  first  heated  and  then  mixed  with  a  small 
quantity  of  fresh  normal  serum  (complement)  and 
with  an  emulsion  of  the  bacterium  whose  amboceptors 
it  is  desired  to  discover.  After  standing  for  six  hours 
at  room  temperature,  red  blood  cells  previously  treated 
with  heated  haemolytic  serum  are  added.  If  there  is 
no  hsemolysis  it  is  held  to  mean  that  the  complement 
in  the  fresh  serum  which  was  suitable  for  lysis  of 
properly  prepared  blood  corpuscles,  has  been  absorbed 
by  the  bacteria  by  reason  of  the  presence  of  specific 
amboceptors  in  the  serum  tested. 

Wassermann  1  has  attempted  to  apply  this  method 
in  measuring  the  amboceptor  content  of  specific  menin- 

*  Wassermann,  Neisser  and  Brack,  Deutsche  med.  Wochen- 
schr.,  1906;  Wassermann  and  Plaut,  Ibid. 


BACTERIOLYSINS   AND  H&MOLYS1XS         73 

gococcus  sera  and  has  successfully  adapted  the  Bordet- 
Gengou  test  to  the  diagnosis  of  syphilitic  infections.1 

Neisser  and  Sachs2  have  recently  described  a  procedure 
for  the  forensic  diagnosis  of  blood  stains.  The  principle  of 
this  is  the  same  as  in  the  preceding,  although  in  so  far  as 
a  specific  precipitin  serum  is  made  use  of,  the  procedure  is 
really  modelled  after  the  "Gengou-Gay"  phenomenon. 

If  human  blood  serum  is  mixed  with  a  specific 
human  precipitin  serum  derived  from  rabbits,  it  will 
be  found  that  the  mixture  binds  complement.  Hae- 
molysin  subsequently  added  is  unable  to  dissolve  its 
specific  red  blood  cells,  owing  to  this  locking  up  of 
the  complement.  Only  the  serum  of  monkeys  has  a 
similar  effect.  The  amount  required  is  extremely 
minute,  TW^o  to  TTy<yV<y<T  cc-  human  blood  or  monkey 
blood  sufficing.  Extracts  of  human  blood  stains  will  also 
produce  the  desired  effect.  The  authors  believe  that 
the  immunization  with  human  blood  serum  gives  rise 
not  only  to  precipitins  but  also  to  amboceptors  which 
then  are  able  to  unite  with  their  corresponding  unformed 
albuminous  bodies  and  so  bind  complement.  Others 
are  of  the  opinion  that  the  complement  is  bound  by 
the  precipitin-precipitum  combination. 

The  test  is  extremely  delicate  and  has  been  found 
trustworthy  by  a  number  of  investigators.  In  view 
of  the  importance  of  such  tests  in  medico-legal  cases, 
Neisser  and  Sachs  suggest  that  it  should  always  be 
used  in  addition  to  the  well  known  Wassermann- 
Uhlenhuth  precipitin  test. 

Normal  Serum,  its  Haemolytic  and  Bacteriolytic 
Action.  —  Inquiring  now  into  the  essential  differ- 

1  See  also  page  185. 
.    2  Neisser  and  Sachs,  Berliner  klin.  Wochenschrift,  1905. 


74 


IMMUNE  SERA 


ence  between  a  specific  haemolytic  or  bactericidal 
serum  and  a  normal  one,  we  must  first  of  all  study 
the  behavior  of  normal  serum  toward  alien  red 
cells  and  bacteria.  It  has  long  been  known  to 
physiologists  that  fresh  normal  serum  of  many 
animals  has  the  power  to  dissolve  blood  cells  of 
another  species.  This  was  studied  especially  by 
Landois.  One-half  to  one  c.c.  of  normal  goat  serum, 
for  example,  is  able  to  dissolve  5  c.c.  of  a  5%  mix- 
ture (in  normal  salt  solution)  of  rabbit  or  guinea 
pig  red  cells.  In  the  same  way,  these  red  cells 
are  dissolved  by  the  sera  of  oxen,  of  dogs,  etc. 
This  normal  globulicidal  property  of  the  serum  cor- 
responds to  another  which  fresh  normal  serum  was 
found  to  possess,  namely,  the  property  to  dissolve 
appreciable  quantities  of  many  species  of  bacteria. 
This  analogy  was  pointed  out  by  Fodor,  Nutall, 
Nissen,  and  especially  by  Buchner.  We  call  this 
the  bactericidal  property  of  fresh  normal  serum. 

This   property   is   well   illustrated   by    the   following 
protocol  from  Park. 


No.  of  bacteria 
in  i  cc.  fluid. 

Amount  of 
serum  added. 

Approximate  number  alive  after  being  kept  at  37°  O 

One  hour. 

Two  hours. 

Twenty-seven  hrs. 

30,000 
100,000 

1,000,000 

0  .  I   CC. 
0  .  I   CC. 
0.  I   CC. 

400 
5,000 
400,000 

2 
1,000 
2,OOO,OOO 

0 
200,000 
10,000,000 

It  is  at  once  apparent  that  the  number  of  bacteria 
introduced  is  an  important  factor,  the  normal  serum 
being  able  to  kill  off  only  a  certain  number. 


BACTERIOLYSINS  AND  H^MOLYSINS  7- 

Buchner,  as  we  have  already  seen,  had  studied 
this  bactericidal  action  carefully  and  ascribed  the 
action  to  a  substance  found  in  all  normal  serum, 
which  he  called  alexin.  According  to  his  experi- 
ments, this  is  a  very  unstable  substance,  decom- 
posing spontaneously  on  standing  or  on  heating  for 
a  few  minutes  to  55°  C.,  or  readily  on  the  action 
of  chemicals.  According  to  this  author  all  the 
globulicidal  and  bactericidal  functions  of  normal 
serum  are  performed  by  this  one  substance,  the 
alexin. 

Active  and  Inactive  Normal  Serum. — In  taking 
up  the  study  of  the  haemolytic  action  of  normal 
serum  Ehrlich  and  Morgenroth  sought  par- 
ticularly to  discover  whether  in  normal  serum 
the  haemolytic  property  depended  on  the  action  of 
a  single  substance,  the  complement  (Buchner's 
alexin),  or  whether  here  as  in  the  specific  haemo- 
lytic serum  it  depended  on  the  combined  action 
of  two  substances.  For  this  purpose  they  used 
guinea-pig  blood,  which  is  dissolved  by  normal 
dog  serum.  If  this  serum  was  heated  to  55°  C.,  it 
lost  its  haemolytic  power.  It  was  necessary  now 
to  show  that  in  this  inactive  dog  serum  there 
remained  a  second  substance  which  could  be  reacti- 
vated after  the  manner  of  reactivating  an  old 
specific  haemolytic  serum.  This  had  its  difficulties, 
for  they  could  not  add  normal  dog  serum.  This, 
as  we  saw,  is  already  haemolytic  for  guinea-pig 


7 6  IMMUNE  SERA 

blood.  "  Possibly/'  said  they,  "  there  exists  a  com- 
plement of  another  animal  which  will  fit  the  hypo- 
thetical second  substance  of  this  dog  serum." 
This  proved  to  be  the  case,  the  complement  of 
guinea-pig  blood  fulfilling  the  requirements.  If 
they  added  to  the  inactive  normal  dog  serum  about 
2  c.c.  normal  guinea-pig  serum  the  haemolytic  prop- 
erty was  restored  and  the  guinea-pig  red  cells 
dissolved  completely.  According  to  Ehrlich,  this  can 
only  be  explained  by  assuming  that  in  guinea-pig 
blood  there  exists  a  complement  which  happens  to  fit 
the  haptophore  group  of  the  second  substance  or 
inter- body,  of  the  normal  dog  serum.  This  com- 
bination of  guinea-pig  blood,  inactive  normal  dog 
serum,  and  a  reactivating  normal  guinea-pig  serum 
is  well  adapted  to  demonstrate  the  existence  in 
normal  dog  serum  of  an  inter-body;  for  the  guinea- 
pig  serum  should  be  the  best  possible  preservative 
for  the  guinea-pig  red  cells.  The  haemolysis  fol- 
lowing the  addition  of  this  serum  shows  positively 
the  existence  of  a  substance  in  the  dog  serum  which 
has  acted  with  something  in  the  guinea-pig  serum.1 

1  Of  such  combinations,  i.e.,  combinations  in  which  a  com- 
plement derived  from  the  same  animal  from  which  the  red  cells 
are  derived  fits  to  the  inter-body  of  other  species  of  animals, 
causing  the  solution  of  red  cells  of  the  latter,  Ehrlich  and 
Morgenroth  found  still  other  examples.  For  instance,  guinea- 
pig  blood,  inactive  calf  serum,  guinea-pig  serum;  goat  blood, 
inactive  rabbit  blood,  goat  serum;  sheep  blood,  inactive  rabbit 
blood,  sheep  serum;  guinea-pig  blood,  inactive  sheep  serum, 
guinea-pig  serum. 


BACTERIOLYSINS  AND  H&MOLYSINS          77 

Inter-body  and  Complement.  —  We  see,  then,  that 
the  haemolytic  action  of  normal  sera  depends,  just 
as  that  of  the  specific  haemolytic  sera,  on  the  com- 
bined action  of  two  bodies:  one,  the  inter-body, 
which  corresponds  to  the  immune  body  of  the 
specific  sera,  and  a  second  or  complement.  In 
speaking  of  the  constituents  of  normal  serum, 
Ehrlich  and  Morgenroth  prefer  to  use  this  term 
inter-body  to  distinguish  it  from  the  immune  bodies 
of  specific  hasmolytic  sera. 

Action  not  Entirely  Specific.  —  It  has  also  been 
found  that  there  frequently  exist  normal  sera  which 
are  haemolytic  not  only  for  one  species  of  red  cell, 
but  for  several.  We  saw,  for  instance,  that  normal 
goat  serum  dissolved  the  red  cells  of  guinea  pigs 
and  rabbits.  The  question  now  arises,  Is  this  prop- 
erty of  normal  goat  serum  due  to  two  inter-bodies 
existing  in  the  serum  side  by  side,  one  fitting  the 
red  cells  of  the  guinea  pig,  the  other  those  of  the 
rabbit?  Ehrlich  and  Morgenroth  answered  this 
in  the  affirmative,  for  in  the  following  experi- 
ment they  succeeded  in  having  each  of  the  two 
inter-bodies  combine  with  its  respective  cell.  To 
some  inactive  normal  goat  serum  they  added  rab- 
bit blood  and  centrifuged  the  mixture.  To  the 
separated  clear  fluid  they  again  added  some  rab- 
bit red  cells  as  well  as  normal  horse  serum  to  reac- 
tivate the  mixture.  Horse  serum  is  not  haemo- 
lytic for  rabbit  red  cells.  The  mixture  remained 


7  8  IMMUNE  SERA 

unchanged,  no  haemolysis  taking  place.  If,  how- 
ever, they  added  some  of  this  normal  horse  serum 
to  the  centrifuged  red  cells,  the  latter  immediately 
dissolved.  Now,  to  the  clear  centrifuged  fluid, 
which  as  we  have  seen  would  not  dissolve  rabbit 
red  cells,  they  added  guinea-pig  red  cells  and  again 
some  normal  horse  serum  to  reactivate  the  mixture. 
The  guinea-pig  red  cells  all  dissolved.  This  proved 
conclusively  that  in  the  normal  goat  serum  there 
had  existed  two  specific  inter-bodies.  One,  for 
rabbit  red  cells,  had  been  tied  by  these  cells  and 
carried  down  with  them  in  centrifuging ;  the  other, 
specific  for  guinea-pig  red  cells,  had  remained 
behind. 

Multiplicity  of  the  Active  Substances.  Further 
investigation  led  these  authors  to  assume  a  still 
greater  multiplicity  in  the  substances  in  normal 
serum  which  are  concerned  in  haemolysis.  In 
addition  to  the  two  interbodies  just  mentioned,  they 
demonstrated  the  existence  in  goat  serum  of  two 
specific  complements,  one  for  each  interbody,  and 
they  were  able  by  means  of  Pukall  filters  to  separate 
these  two.  In  this  filtration  the  complement  fit- 
ting the  inter-body  for  rabbit  blood  remained 
behind  for  the  greater  part,  while  that  fitting 
the  inter-body  for  guinea-pig  blood  mostly  passed 
through. 

Whereas  then,  according  to  Buchner,  only  one 
substance,  the  alexin,  is  concerned  in  the  haemo- 


B ACT ERIOLY SINS   AND   H JEM OLY SINS          79 

lytic  action  of  this  normal  goat  serum,  these 
experiments  of  Ehrlich  and  Morgenroth  show 
us  four  substances,  viz.,  two  inter-bodies  and 
two  complements.  This  at  once  makes  clear  the 
opposing  views  of  these  authorities.  According  to 
Ehrlich,  however,  the  number  of  active  substances 
in  normal  serum  is  still  greater,  for  it  often  hap- 
pens that  a  specific  inter-body  shows  itself  to  be 
made  up  of  several  inter-bodies,  all,  to  be  sure, 
fitting  the  same  specific  red  cell,  but  differing 
from  each  other  by  their  behavior  toward  dif- 
ferent complements.  Ehrlich,  therefore,  regards 
the  substances  concerned  in  haemolysis  which 
occur  in  normal  serum  to  be  of  great  number  and 
variety. 

Difference  between  a  Normal  and  a  Specific 
Immune  Serum.  —  Practical  Application.  —  Return- 
ing now  to  the  question  of  the  difference  between  a 
specific  immune  serum  and  a  normal  one,  we  find 
this  to  be  as  follows :  Normal  serum  contains  a  great 
variety  of  inter-bodies,  in  very  small  amounts,  and  a 
considerable  amount  of  complements.  In  immune 
serum,  on  the  other  hand,  the  amount  of  a  specific 
inter-body,  the  one  which  fits  the  haptophore  group 
of  a  certain  cell,  is  enormously  increased.  This 
specifically  increased  inter-body,  it  will  be  remem- 
bered, is  called  the  immune  body.  The  comple- 
ment, as  shown  by  v.  Dungern,  Bordet,  Ehrlich 
and  Morgenroth  and  Wassermann,  is  in  no  way 


80  IMMUNE  SERA 

increased  by  the  immunizing  process.  The  increase 
affects  solely  the  immune  body.  It  is  therefore 
possible  to  have  a  serum  which  contains  more 
immune  body  than  complement  to  satisfy  it,  and  if 
we  withdraw  such  a  serum  from  an  animal  we  shall 
find  that  it  contains  some  free  immune  body.  This 
serum  can  only  then  exert  its 'full  power  when  the 
full  amount  of  complement  is  present,  i.e.,  when 
some  normal  serum  is  added.  If  we  treat  a  rabbit 
with  the  red  cells  of  an  ox,  as  v.  Dungern  did,  we 
shall  obtain  a  serum  which  is  haemolytic  for  ox 
blood.  Of  this  freshly  drawn  serum  0.05  c.c.  suf- 
fice to  dissolve  5.0  c.c.  of  a  5%  mixture  of  ox 
blood.  If  now  we  add  to  this  haemolytic  serum 
a  little  normal  rabbit  serum,  we  shall  find  that 
only  one-tenth  of  the  amount  of  serum  is  required; 
i.e.,  only  0.005  c-c-  to  dissolve  the  same  quantity 
of  ox  blood.  This  means  that  through  the  addi- 
tion of  the  rabbit  serum,  which,  of  course,  is  not 
haemolytic  for  ox  blood,  a  sufficient  amount  of 
complement  was  added  to  enable  all  the  immune 
body  of  the  specific  serum  to  act.  This  specifically 
increased  power  of  the  immune  serum  to  act  on 
certain  definite  cells  depends  on  the  fact  that  the 
immune  body  resulting  from  the  immunizing 
process  concentrates  the  action  of  the  comple- 
ment scattered  through  the  serum,  on  cells  for 
which  it  has  definite  affinities.  If  2  c.c.  of  normal 
guinea-pig  serum  are  able  to  dissolve,  we  will  say 


BACTERIOLYSINS  AND  H&MOLYSlNS  8l 

5  c.c.  of  a  5%  defibrinated  rabbit-blood  mixture, 
and  if  we  find  that  after  the  immunizing  process 
0.05  c.c.  of  the  guinea-pig  serum  suffice  to  dissolve 
the  same  amount  of  rabbit  blood,  we  conclude 
that  through  this  process  the  inter-body,  i.e.  the 
immune  body,  has  been  increased  forty  times.  We 
know  that  the  complement  has  not  been  increased, 
but  this  is  now  able  to  act  by  means  of  forty  times 
increased  combining  facilities.  This  increase,  how- 
ever, is  exclusively  for  rabbit-blood  cells.  In  a 
bactericidal  immune  serum  this  specific  increase  is 
sometimes  as  much  as  100,000  times  that  of  normal 
serum. 

The  practical  idea  to  be  gained  from  this  for 
the  therapy  of  infectious  diseases  is  this:  that 
with  the  injection  of  an  immune  serum  we  supply 
only  one  of  the  necessary  constituents  to  kill 
and  dissolve  the  bacteria,  and  that  is  the  immune 
body. 

We  do  not,  however,  supply  the  second,  i.e.  the 
complement,  for  this  we  have  seen  is  not  increased 
by  the  immunizing  process.  As  matters  stand, 
then,  the  use  of  a  specific  immune  serum  for 
therapeutic  purposes  assumes  that  the  complement 
which  is  essential  for  the  action  of  the  immune 
body  will  be  found  in  the  organism  to  be  treated. 
Since  in  certain  infectious  diseases  the  required 
complement  is  present  in  too  small  amounts  in 
the  organism,  Wassermann  suggested  that  the 


82  IMMUNE   SERA 

curative  power  of  many  bactericidal  sera  might  be 
increased  by  the  simultaneous  injection  of  the  sera 
of  certain  normal  animals  in  order  thus  to  gain 
an  increased  amount  of  complement;  but  we 
shall  soon  see  that  this  procedure,  while  of  great 
value  in  animal  experiments,  presents  certain  dif- 
ficulties. 

Nature  of  the  Immune  Body  —  Partial  Immune 
Bodies  of  Ehrlich  —  Turning  now  to  a  closer  study  of 
the  nature  of  the  immune  body,  we  again  find  a  dif- 
ference of  opinion.  Whereas  Bordet,  MetchnikofI, 
and  Besredka  assume  each  immune  body  to  be  a 
single  definite  substance,  Ehrlich  and  Morgenroth 
as  a  result  of  their  experiments  hold  to  a  plurality 
of  bodies. 

These  authors  say  that  each  immune  body 
is  built  up  of  a  number  of  partial-immune  bodies, 
a  point  to  which  we  have  already  alluded.  In 
support  of  this  view  they  offer  the  following  ex- 
periment. On  immunizing  a  rabbit  with  ox  blood, 
they  obtained  a  serum  haemolytic  not  only  for 
ox  blood  but  also  for  goat  blood;  on  immunizing 
a  rabbit  with  goat  blood  they  obtained  a  serum 
haemolytic  for  goat  blood  and  ox  blood.1 

The  conditions  present  can  be  readily  under- 
stood by  reference  to  Fig.  7,  which  represents 
schematically  three  portions  of  the  combining  groups 

1  We  have  already  called  attention  to  these  exceptions  to  the 
rule  of  specific  action. 


BACTERIOLYSINS  AND  HAZMOLYSINS  83 

of  the  blood  cells.  Of  these  a  is  present  only  in  the 
ox-blood  cells,  ^  only  in  the  goat-blood  cells,  and  ft 
in  both.  If  a  rabbit  is  injected  with  ox  blood,  the 
immune  bodies  corresponding  to  groups  a  and  ft 
will  be  formed.  On  subjecting  such  a  serum  to 
absorption  with  ox-blood  cells  we  shall  find  that 
these,  by  means  of  their  a  and  ft  groups  will  be  able  to 
absorb  all  the  immune  bodies,  whereas  goat-blood 
cells  will  in  a  similar  test  absorb  only  the  immune 


FIG.  7 


body  of  portion  ft,  leaving  the  immune  body  of 
portion  a  in  solution. 

According  to  Ehrlich's  theory,  then,  the  red  cells 
of  the  ox  possess  certain  receptors  which  are  identi- 
cal with  receptors  possessed  by  the  goat  red  cells. 
From  this  it  follows  that  in  a  single  red  cell  there 
are  several  or  many  groups  each  of  which  is  able, 
when  it  finds  a  fitting  receptor,  to  take  hold  of  a 


84  IMMUNE  SERA 

single  immune  body.  Ehrlich  and  Morgenroth, 
therefore,  claim  that  the  immune  body  of  a  haemo- 
lytic  serum  is  composed  of  the  sum  of  the  partial 
immune  bodies  which  correspond  to  the  individual 
receptors  used  to  excite  the  immunity.  It  may  be 
assumed,  then,  that  not  all  of  the  combining  groups 
of  a  cell,  be  this  a  blood  cell  or  a  bacterium,  will 
find  fitting  receptors  in  every  animal  organism, 
and  that  therefore  not  all  the  possible  partial  im- 
mune bodies  will  be  equally  developed.  In  one 
animal  there  may  be  receptors  which  are  not  pres- 
ent in  another,  and  in  this  way  there  might  be  a  dif- 
ferent variety  of  partial  immune  bodies  in  the  two 
animals.  This  would  lead  to  the  possibility  of  the 
occurrence  of  immune  bodies,  for  the  same  species 
of  blood  cell  or  bacterium,  differing  from  each  other 
in  the  partial  immune  bodies  composing  them, 
according  to  the  variety  of  animals  used  in  prepar- 
ing the  serum. 

Metchnikoff's  Views  —  Practical  Importance  of 
the  Point.  —  This  view  is  directly  opposed  to  that  of 
MetchnikofT  and  Besredka,  who  believe  that  a  cer- 
tain immune  body,  e.g.  one  specific  for  ox  blood, 
is  always  the  same  no  matter  from  what  animal  it 
is  derived.  The  point  is  not  merely  theoretical, 
but  under  certain  circumstances  of  great  practical 
importance.  If  we  believe,  as  Ehrlich  does,  that 
the  immune  body  differs  according  to  the  species  of 
animal  from  which  it  is  derived,  i.e.,  that  it  is  made 


BACTERIOLYSINS  AND  H&MOLYSINS  85 

up  of  different  partial-immune  bodies,  then  we  must 
admit  that  we  have  better  chances  for  finding  fit- 
ting complements  if  we  make  use  of  immune  bodies 
derived  from  a  variety  of  animals.  We  would,  for 
instance,  be  likely  to  achieve  better  results  in  treat- 
ing a  typhoid  patient  with  a  mixture  of  specific 
bactericidal  typhoid  sera  derived  from  a  variety  of 
animals  than  if  we  used  a  serum  derived  only  from 
a  horse.  For  in  such  a  mixture  of  immune  bodies 
the  variety  of  partial-immune  bodies  must  be  very 
great  and  the  chances  that  the  complements  of  the 
human  body  will  find  fitting  immune  bodies,  and  so 
lead  to  the  destruction  of  the  typhoid  bacilli,  are 
greatly  increased.  Ehrlich  and  his  pupils  have 
actually  proposed  such  a  procedure  in  the  use  of 
bactericidal  sera  for  therapeutic  purposes.1 

Support  for  Ehrlich' s  View.  —  Besides  the  above 
experiments  we  possess  others  which  support  the 
theory  that  the  immune  body  is  not  a  simple  but 
a  compound  substance,  v.  Dungern  had  already 
shown  that  following  the  treatment  of  an  animal 
with  ciliated  epithelium  from  the  trachea  of  an  ox, 
there  were  developed  immune  bodies  which  acted 
not  only  on  the  ciliated  epithelium  but  also  on  the 
red  cells  of  oxen.  We  must  assume,  therefore,  that 

1  Reasoning  along  similar  lines,  namely,  that  the  human 
complement  must  fit  the  immune  body  of  the  therapeutic 
serum,  Ehrlich  has  also  proposed  that  these  bactericidal  sera 
be  derived  from  animals  very  closely  related  to  man,  e.g., 
apes,  etc. 


86  IMMUNE  SERA 

the  ciliated  epithelium  and  the  red  cells  of  the  ox 
possess  common  receptors.  Analogous  to  this  is 
the  action  of  the  immune  body  resulting  from  the 
injection  of  spermatozoa,  as  was  pointed  out  by 
Metchnikoff  and  Moxter. 

We  see,  then,  that  the  specific  action  of  immune 
bodies  is  not  so  limited  as  to  apply  only  to  the  cells 
used  in  the  immunizing  process,  but  extends  to 
other  cells  which  have  receptors  in  common  with 
these." 

So  far  as  concerns  the  site  in  the  organism  where 
the  substances  used  in  immunizing  find  their 
receptors,  this  is  not  known  for  the  hsemolytic 
immune  body.  For  the  bactericidal  immune  bodies 
of  cholera  and  typhoid  the  researches  of  Pfeiffer, 
Marx,  and  others  show  that  the  chief  site  of  pro- 
duction is  in  the  bone-marrow,  spleen,  and  lymph 
bodies.  Wassermann's  experiments  on  local  immu- 
nity indicate  that  the  site  of  infection  determines 
largely  the  site  of  the  development  of  the  immune 
bodies. 

Antihaemolysins :  their  Nature  —  Anti-complement 
or  Anti-immune  Body?  —  A  further  step  in  the 
study  of  haemolysins  is  one  discovered  independ- 

1  The  same  holds  good  for  the  agglutinins  and  the  pre- 
cipitins  still  to  be  studied.  In  these  the  action  extends  also 
to  closely  related  cells  and  bacteria,  or  in  the  case  of  the  precipi- 
tins  to  closely  related  albumins,  as  these  possess  a  number  of 
receptors  which  are  common  to  them  and  to  the  cells  or  sub- 
stances used  for  immunizing. 


BACTERIOLYSINS  AND  H^EMOLYSINS  87 

ently  by  Ehrlich  and  Morgenroth  on  the  one  hand, 
and  Bordet  on  the  other.  These  authors  succeeded 
in  producing  an  antih&molysin.  The  procedure  is 
closely  related  to  the  results  gained  by  immuniza- 
tion against  bacterial  poisons.  A  specific  haemoly- 
sin,  one,  for  example,  specific  for  rabbit  blood, 
derived  by  treating  a  guinea  pig  with  rabbit  red 
cells,  is  highly  toxic  to  rabbits.  Injected  into  the 
animals  intravenously  in  doses  of  5  c.c.  it  kills  the 
animals  acutely,  causing  intra  vitam  a  solution  of 
the  red  cells.  Such  a  haemolytic  serum,  then,  acts 
the  same  as  a  bacterial  poison,  and  it  is  possible  to 
immunize  against  this  just  as  well  as  against  a  bac- 
terial poison.  For  example,  to  keep  to  our  illustra- 
tion, rabbits  are  injected  first  with  very  small  doses 
of  this  specific  haemolytic  serum.  The  dose  is 
gradually  increased  until  it  is  found  that  the  animal 
tolerates  amounts  that  would  be  absolutely  fatal  to 
animals  not  so  treated.  If  some  of  the  serum  of 
this  animal  is  now  abstracted  and  added  to  the 
specific  haemolytic  serum,  it  is  found  that  the  power 
of  the  latter  will  be  inhibited.  According  to 
Ehrlich  an  antihanwlysin  has  been  formed.  As 
we  know  that  the  action  of  the  haemolysin  depends 
on  the  combined  action  of  two  substances,  the 
immune  body  and  the  complement,  the  question 
arises  to  which  of  these  two  the  antihasmolysin  is 
related.  Is  it  an  anti-immune  body  or  an  anti- 
complement?  A  study  of  this  question  shows  that 


88  IMMUNE   SERA 

both  these  substances  are  apparently  present.  In 
the  serum  of  the  rabbit  treated  with  specific  haemo- 
lysin,  both  an  anti-immune  body  and  an  anti- 
complement  have  been  found.  Ehrlich  and  Mor- 
genroth  were  further  able  to  show  that  the  action 
of  the  anti-complement  depended  on  a  haptophore 
group  which  it  possessed,  enabling  it  to  combine 
with  the  haptophore  group  of  the  complement, 
thus  satisfying  this  and  hindering  its  combination 
with  the  complementophile  group  of  the  immune 
body. 

"  Anti- complement."  l — *Since  the  complements  are 
constituents  of  normal  serum,  it  should  be  possible 
to  produce  anti-complements  by  injecting  animals 
merely  with  normal  serum;  and  they  can,  in  fact, 
be  so  produced.  If  rabbits  are  treated  by  inject- 
ing them  several  times  with  normal  guinea  pig 
serum,  a  serum  may  be  obtained  from  these  rabbits 
which  contains  anti-complements  against  the  com- 
plements of  normal  guinea-pig  serum.  A  serum 
obtained  in  this  way  of  course  contains  only  one  of 
the  antihaemolytic  bodies,  the  anticomplement, 
and  not  the  antiimmune  body.  This  is  because 
normal  serum  is  too  poor  in  immune  body  (inter- 
body)  to  excite  the  production  of  any  antiimmune 
body. 

1  The  existence  of  anti-complements  is  denied  by  Bordet, 
Gay  and  others.  For  a  statement  of  their  views  see  pa<je 
96. 


BACTERIOLYSINS  AND  IL-EMOLYSINS          89 

If  to  a  hsemolytic  serum  derived  from  guinea  pigs 
we  add  an  anticomplement  serum  derived,  as  just 
stated,  from  rabbits,  and  containing  an  anticom- 


*  I 


ii. 

f 

COMPLEMENT 


COMPLEMENT 

ANTICOMPLEMENT 


IMMUNE    BODY  J|  f        IMMUNEBODY 

CELL   MB  m    m  CELL 

FIG.  8.     (After  Levaditi.) 

plement  specific  for  guinea-pig  complement,  the 
haemolytic  action  of  the  former  will  be  inhibited,  for 
the  reason  that  the  complement  necessary  for  the 
haemolysis  to  take  place  has  been  bound  by  the 
anticomplement.  (See  Fig.  8.)  One  must,  how- 
ever, observe  the  precaution  to  heat  the  anticom- 
plement serum  of  the  rabbit  to  55°C.  before  so 
mixing  it,  in  order  to  destroy  the  complement  which 
it  contains  and  which  would  otherwise  reactivate  the 
guinea-pig  immune  body. 

From  the  foregoing  we  see  that  either  anti- 
immune  body  alone,  or  anticomplement  alone,  is 
able  to  inhibit  the  hxmolytic  action.  Haemoly- 
sis cannot  take  place  when  either  of  the  two 


9° 


IMMUNE   SERA 


necessary  factors  is  bound  and  prevented  from 
acting. l 

The  action  of  anticomplements  is  specific,  i.e.,  an 
anticomplement  combines  only  with  its  specific 
complement.  Thus  an  anticomplement  serum 
derived  from  rabbits  by  treatment  with  guinea- 
pig  serum  combines  only  with  the  complement  of 
normal  guinea-pig  serum,  not,  however,  with  the 
complements  of  other  animals.  Exceptions  to  this 
are  those  cases  in  which  the  complement  of  the 
other  species  possess  receptors  identical  with  those 
of  the  first. 

In  order  that  a  normal  serum  of  species  A, 
injected  into  species  B,  produce  anticomplements 
there,  the  side-chain  theory  demands  that  the  com- 
plements of  A  find  fitting  receptors  in  species  B. 
According  to  Ehrlich,  however,  normal  serum  con- 
tains many  different  complements  and  not  merely  a 
single  one.  Under  the  circumstances,  it  is  easily 
possible  that  only  a  few  of  the  complements  in  the 
serum  of  A  find  fitting  receptors  in  species  B.  We 
shall  then  obtain  an  anticomplement  serum  which 
inhibits  the  action  of  some,  but  not  of  all  the  com- 
plements of  species  A.  Thus  it  might  inhibit  the 

1  By  treating  animals  with  normal  sera  of  certain  other 
species,  it  is  possible  to  produce  not  only  anti-complements, 
but  also  specific  anti-bodies  against  certain  other  constituents 
of  normal  serum.  These  are,  for  example,  anti-agglutinins, 
which  inhibit  the  action  of  the  haemagglutinins  of  normal  serum, 
and  anti-precipitins,  which  we  shall  discuss  later. 


BACTERIOLYSINS    AND   HsEMOLYSINS        91 

action  of  a  complement  fitting  to  a  certain  bacteri- 
cidal immune  body  and  not  of  one  contained  in  the 
same  serum  which  fitted  a  certain  haemolytic  im- 
mune body,  etc. 

Fluctuations  in  the  Amount  of  Complement  and 
other  Active  Substances  in  the  Blood — We  have 
come  to  know  certain  conditions  under  which  there 
may  be  a  decrease  of  certain  complements  in 
normal  serum.  Ehrlich  and  Morgenroth  showed 
that  in  rabbits  poisoned  with  phosphorus  and  in 
whom,  therefore,  the  liver  was  badly  damaged^ 
the  serum  on  the  second  day  (the  height  of  the 
disease)  had  lost  its  power  to  dissolve  guinea-pig 
blood,  and  that  this  was  due  to  a  disappearance  of 
the  complement.  Metchnikoff  also  reported  that  in 
an  animal  suffering  from  a  continuing  suppurating 
process,  the  complement  had  fallen  considerably  in 
amount.  Especially  interesting  are  the  experi- 
ments of  v.  Dungern,  who  showed  that  animal  cells, 
hence  emulsions  of  fresh  organs,  are  able  to  attract 
and  combine  with  complements. 

Even  more  important  than  the  question  of  a 
decrease  in  complements,  or  an  inhibition  of  their 
action,  is  that  of  the  possibility  to  artificially  in- 
crease them.  A  number  of  authors,  among  them 
Nolf  and  Miiller,  have  answered  this  question  in  the 
affirmative.  They  believe  they  have  noticed  such 
an  increase  following  the  injection  of  an  animal  with 


92  IMMUNE  SERA 

all  sorts  of  substances,  such  as  normal  serum  of 
another  animal,  sterile  bouillon,  etc.  v.  Dungern, 
Wassermann  and  others,  have  not  been  able  to  con- 
vince themselves  of  the  possibility  of  such  a  definite 
increase.  Wassermann  tried  to  excite  the  increased 
production  of  complement  by  injecting  guinea  pigs 
for  some  time  with  anticomplement.  This  being 
the  opposite  of  the  complement,  he  hoped  to  be 
able  by  immunizing  to  excite  an  increase  of  the 
complements.  In  this  he  was  unsuccessful,  though 
of  course  it  maybe  possible  with  another  species  of 
animal. 

Despite  all  this,  we  must  believe  that  the  amount 
of  complement,  as  well  as  the  amount  of  other  active 
substances  of  the  blood,  inter-bodies,  agglutinins, 
antitoxins,  ferments,  antiferments,  etc.,  is  subject 
to  great  fluctuations  even  in  the  same  individual, 
a  constant  change  going  on  within  the  organism. 
Ehrlich,  in  particular,  has  pointed  out  these  indi- 
vidual and  periodic  variations  and  has  insisted  on 
their  importance.  Very  likely,  under  circumstances 
of  which  we  now  know  very  little,  these  substances 
are  at  certain  times  produced  in  greater  amounts, 
at  other  times  in  lesser;  sometimes  they  may  be 
absent  entirely  in  an  individual  in  whom  they  were 
previously  present.  For  example,  the  serum  of  a 
dog  will  at  times  dissolve  the  red  cells  of  cats,  rab- 
bits, and  guinea  pigs,  at  other  times  not.  Further- 
more, the  serum  of  one  and  the  same  animal  may 


B ACT ERIOLY SINS  AND  H.EMOLYSINS  p* 

possess  specific  haemolytic  properties  for  certain 
cells,  and  later  on  may  lose  this  property  entirely. 
In  human  serum  these  same  individual  and  periodic 
variations  may  be  demonstrated,  as  Wassermann 
was  able  to  prove  experimentally.  However,  the 
circumstances  on  which  these  variations  depend  are 
as  yet  entirely  unknown  to  us.  Possibly  we  are 
dealing  here  with  subtle  pathological  changes. 

Source  of  the  Complements  —  Leucocytes  as  a 
Source  —  Other  Sources.  —  Where  do  the  comple- 
ments or  alexins  originate?  This  question  has  been 
studied  particularly  by  Metchnikoff  and  by  Buch- 
ner;  also  by  Bail,  Hahn,  Schattenfroh,  and  others. 
These  investigators  believe  that  the  leucocytes  are 
the  source  of  the  complements  or  alexins.  There 
is,  however,  this  difference  between  the  views 
of  Metchnikoff  and  Buchner:  whereas  Buchner 
believes  the  alexins  to  be  true  secretory  products, 
Metchnikoff  believes  that  they  originate  on  the 
breaking  up  of  the  leucocytes,  i.e.,  that  they  are  de- 
composition products.  Metchnikoff  bases  his  belief 
chiefly  on  the  work  of  his  pupil,  Gengou,  who  showed 
that  although  the  serum  was  rich  in  alexin  (i.e.,  com- 
plement) the  plasma  contained  none  at  all. 

Other  authors,  as  Pfeiffer  and  Moxter,  as  a  result 
of  their  experiments,  are  not  willing  to  assume  the 
existence  of  any  relationship  between  the  alexins 
and  the  leucocytes.  Gruber  as  well  as  Schatten- 
froh are  ready  to  believe  the  leucocytes  to  be  the 


94  IMMUNE  SERA 

source  of  an  alexin,  but  claim  that  this  is  different 
from  that  found  in  serum.  Wassermann  believes 
that  the  leucocytes  are  a  source  of  complements 
(alexins),  for  he  succeeded  in  producing  anticom- 
plement  by  means  of  injections  of  pure  leucocytes 
which  had  been  washed  free  from  all  traces  of  serum, 
and  which  had  been  obtained  by  injections  of  aleu- 
ronat.  In  view  of  the  plurality  of  the  comple- 
ments, Wassermann  expressed  the  view  that  the 
leucocytes  are  probably  one  source,  but  not  the 
only  one,  for  the  complements  of  the  serum.  Land- 
steiner  and  Donath  have  confirmed  this  experi- 
mentally. They  succeeded  in  producing  anticom- 
plement  by  the  injection,  not  only  of  leucocytes, 
but  of  other  animal  cells.  Furthermore,  the  experi- 
ments of  Ehrlich  and  Morgenroth  already  mentioned, 
in  which  the  complements  disappeared  after  the 
destruction  of  the  liver  function,  show  that  the  liver 
cells  are  concerned  in  the  formation  of  complements. 
Structure  of  Complements — Haptophore  and  Zy- 
motoxic  Groups — Complementoids. — Ehrlich 's  con- 
ception of  the  structure  of  complements  is  based 
principally  on  the  fact  that  when  complement  is 
heated  to  55°  C.,  its  complementing  power  is  lost. 
If  animals  are  injected  with  such  a  heated  serum, 
anticomplement  will  be  produced,  showing,  accord- 
ing to  Ehrlich,  that  heating  has  not  destroyed  the 
entire  complement  body,  but  only  that  part  which 
effects  the  digesting,  solvent  action.  The  hapto- 


BACTERIOLYSINS  AND  HMMOLYSINS 


95 


phore  group  must  have  remained  intact.  Ehrlich 
therefore  concludes  that  the  complements  consist 
of  a  combining  haptophore  group  which  withstands 
heating  to  55°  C.,  and  another,  more  fragile  group, 
which  possesses  the  actual  solvent  properties  and 
which  Ehrlich  calls  the  zymotoxic  group.  The 
complements,  according  to  this  conception,  are 
entirely  analogous  to  the  toxins  already  studied. 

zymotoxic  group 
COMPLEMENT 
haptophore  group 

IMMUNE  BODY 

FIG.  9. 

And  just  as  those  toxins  which  had  lost  their 
toxophore  group  were  called  toxoids,  so  Ehrlich 
terms  complements  which  have  lost  their  zymo- 
toxic group,  complementoids . 

Bordet,  it  will  be  recalled,  does  not  believe  in 
any  complementophile  group  in  the  amboceptor. 
So  also  in  the  case  of  the  complement,  or  alexin 
as  he  prefers  to  call  it,  he  refuses  to  accept  Ehrlich 's 
view  that  this  contains  a  zymotoxic  and  a  hapto- 


96  IMMUNE  SERA 

phore  group.  The  fact  that  heated  complement 
produces  an  anticomplementary  serum  is  readily 
explained  by  Bordet  as  due  to  the  absorption  of 
complement  by  precipitates.  An  illustration  will 
bring  out  this  point  more  clearly. 

Goat  serum  heated  to  55°  C.,  and  therefore  con- 
taining no  active  complement,  is  injected  into  a 
rabbit.  According  to  Ehrlich  it  excites  the  pro- 
duction of  an  "  anticomplement  "  in  response  to 
the  "  complementoid  "  which  it  contains.  As  we 
shall  see  in  the  next  chapter,  the  injection  of  this 
heated  goat  serum  in  addition  to  whatever  else  it  may 
do  does  actually  excite  the  production  of  a  specific 
precipitin  in  the  serum  of  the  rabbit,  so  that  when 
such  a  rabbit  serum  is  mixed  with  goat  serum,  a  pre- 
cipitate will  be  "produced.  Ehrlich 's  demonstration 
of  the  "  anticomplement  "  is  somewhat  as  follows: 
Ox  blood  corpuscles,  plus  suitable  amboceptor  (serum 
of  a  rabbit  injected  with  ox  blood  corpuscles),  plus 
fresh,  normal  goat  serum  as  complement,  undergo 
haemolysis.  When,  however,  the  fresh,  normal 
goat  serum  is  mixed  with  the  serum  of  a  rabbit 
previously  injected  with  goat  serum,  and  then  the 
above  combination  carried  out,  no  haemolysis 
occurs.  The  rabbit  serum  contains  an  "  anti- 
complement,"  says  Ehrlich.  On  the  other  hand, 
Bordet  and  Gay  believe  that  the  anticomplement- 
ary action  is  due  to  the  absorption  of  goat  com- 
plement by  the  precipitate  produced  by  the  mix- 


BACTERIOLYSINS  AND  H.&MOLYSINS 


97 


ture  of  goat  serum  and  its  precipitin,  the  rabbit 
serum.  Moreschi  comes  to  the  same  conclusion. 

Isolysins  —  Autolysins  —  Anti-isolysins.  —  All  of 
the  preceding  studies  in  haemolysis  have  concerned 
themselves  with  the  results  obtained  by  injecting 
animals  of  one  species  with  blood  cells  of  another. 
Ehrlich  and  Morgenroth  now  sought  to  discover 
what  the  results  would  be  if  they  injected  an  animal 
with  blood  cells  of  its  own  species.  They  injected 
goats  with  goat  blood,  and  found  that  when  the 
amount  injected  at  one  time  was  large  the  serum 
of  the  goat  injected  acquired  hasmolytic  properties 
for  the  blood  of  many  other  goats,  but  not  for  all. 
The  substances  thus  formed  the  authors  called 
isolysins.  These,  then,  are  substances  which  will 
dissolve  the  blood  of  other  individuals  of  the  same 
species.  Substances  which  dissolve  the  blood  cells 
of  the  same  individual  are  called  autolysins.  But 
autolysins  have  so  far  been  demonstrated  experi- 
mentally only  once  (by  Ehrlich  and  Morgenroth). 
If  one  tests  the  properties  of  an  isolysin  of  a  goat  on 
the  blood  of  a  great  many  other  goats,  it  will  be 
found  that  this  will  be  strongly  solvent  for  the 
blood  of  some,  slightly  for  the  blood  of  others,  and 
not  at  all  for  still  others. 

By  using  a  blood  that  was  readily  dissolved  by 
the  isolysin,  and  proceeding  in  the  same  series  of 
experiments  which  we  have  already  studied  under 
haemolysis,  Ehrlich  and  Morgenroth  showed  that 


98  IMMUNE  SERA 

the  isolysins,  like  the  hsemolysins,  consist  of  an 
immune  body  and  a  complement  of  the  normal 
serum.  The  experiments  undertaken  by  these 
authors  were  made  on  thirteen  goats,  and  the  sur- 
prising fact  developed  that  the  thirteen  resulting 
isolysins  were  all  different.  For  example,  the  iso- 
haemolytic  serum  of  one  goat  dissolved  the  red  cells 
of  goats  A  and  B\  that  of  a  second  goat  those  of 
C  and  D ;  of-  a  third  those  of  A  and  D,  but  not  of  C, 
and  so  on.  If  now  they  produced  antiisolysins  by 
injecting  animals  with  these  isolysins,  they  found 
that  these  antiisolysins  were  specific;  i.e.,  the  anti- 
isolysin  of  A  would  inhibit  the  action  only  of  iso- 
lysin  of  A,  but  not  of  C,  etc.  These  results  are  of 
the  highest  clinical  interest,  for  they  show  a  differ- 
ence in  similar  cells  of  the  same  species,  something 
that  had  never  before  been  suspected.  In  the  above, 
the  blood  cells  of  species  A  must  have  a  different  bi- 
ological constitution  than  those  of  species  C,  etc. 

Moss  finds  that  isolysins  occur  in  about  2  5  %  of  adult 
human  individuals,  and  that  the  relative  frequency  is 
the  same  in  health  and  disease,  so  that  the  presence 
of  isolysins  has  no  diagnostic  significance.  The  sub- 
ject has  recently  acquired  importance  because  of  the 
revival  of  homologous  transfusion,  and  it  is  customary 
now  to  always  test  the  blood  of  both  donor  and  re- 
cipient prior  to  carrying  out  such  a  transfusion.  The 
technique  of  the  test  is  described  in  the  appendix. 

The  fact  that  after  injections  of  large  amounts  of 


BACTER10LYSINS  AND   HMMOLYSINS 


99 


cells  of  the  same  species  isolysins  develop,  but  that 
autolysins  are  almost  never  formed,  caused  Ehr- 
lich  and  Morgenroth  to  assume  that  the  body  pos- 
sesses distinct  regulating  functions  which  naturally 
prevent  the  formation  of  the  highly  destructive 
autolytic  substance.  It  is  •  obvious  that  if  there 
were  no  such  regulating  facilities,  the  absorption  of 
large  bloody  effusions  and  hemorrhages  might  lead 
to  the  formation  by  the  organism  of  autolysins 
against  its  own  blood  cells.  Gengou,  a  pupil  of 
Metchnikoff,  believes  to  have  shown  experimen- 
tally that  the  destructive  action  of  these  auto- 
lysins is  hindered  by  the  simultaneous  production 
of  an  auto-antiimmune  body  which  immediately 
inhibits  their  action. 

In  order  that  isolysins  may  be  formed,  it  seems 
necessary  to  overwhelm  the  organism  once  or  sev- 
eral times  with  large  amounts  of  cells  or  cell  prod- 
ucts of  the  same  species ;  to  produce,  as  Ehrlich  says, 
an  ictus  immunisatorius.  Wassermann  tried,  by 
using  various  blood  poisons,  such  as  haemolytic  sera, 
toluylenediamine,  etc.,  for  a  continued  length  of 
time,  to  cause  the  formation  of  these  isolysins,  but 
without  success,  although  in  these  experiments 
each  injection  was  followed  by  an  appreciable 
destruction  of  red  cells  and  absorption  of  their 
decomposition  products.  The  gradual  and  even 
repeated  absorption  of  not  too  large  quantities  of 
decomposed  red  cells  does  not  therefore  lead  to  the 


100  IMMUNE  SERA 

formation  of  isolysins;  but,  as  already  said,  a  sudden 
overwhelming  of  the  organism  by  large  amounts 
of  the  cells  or  their  products  is  necessary. 

Deflection  of  Complement.  —  In  the  use  of  the 
antitoxic  sera,  experience  has  shown  that  the  em- 
ployment of  a  large  dose  is  of  paramount  importance. 
So  far  as  the  antitoxic  action  is  concerned  l  one 
cannot  do  harm  by  giving  a  large  excess.  Con- 
cerning the  action  of  bactericidal  sera,  however, 
the  literature  contains  a  number  of  examples  which 
indicate  that  here  an  excess  of  immune  serum  is 
occasionally  injurious.  Perhaps  the  earliest  proto- 
col of  this  kind  is  that  published  by  Loffler  and 
Abel 2  on  their  experiments  with  bacillus  coli  and  a 
corresponding  immune  serum.  Out  of  nineteen 
guinea  pigs  which  had  been  inoculated  with  the 
same  amount  of  culture  and  had  received  varying 
amounts  of  immune  serum,  only  six  animals  were 
protected,  those  which  had  received  doses  of  0.25 
c.c.  to  0.02  c.c.  Eight  animals  with  larger  doses, 
as  well  as  five  with  smaller  doses  of  serum  died. 
Neisser  and  Wechsberg 3  encountered  the  same 
phenomenon  in  bactericidal  test-tube  experiments, 
and  concluded  as  a  result  of  their  experiments 

1  We  shall  discuss  the  rash  production,  or  "  serum  sickness," 
page  141. 

3  F.  Loffler  and  R.  Abel,  Centralblatt  Bacteriol.,  1896,  Vol. 
xix,  p  51. 

8  M.  Neisser  and  F.  Wechsberg,  Munch,  med.  Wochen- 
schrift.  1901.  No.  1 8. 


BACTERIOLYSINS  AND  H&MOLYS1NS        IOI 


102  IMMUNE  SERA 

that  the  only  satisfactory  explanation  was  one  based 
on  the  views  of  Ehrlich  and  Morgenroth.  In  Fig. 
10,  A  II  represents  schematically  a  bacterium  a 
with  a  number  of  receptors;  for  there  are  many 
reasons  for  assuming  that  each  bacterium  possesses  a 
number  of  receptors  of  the  same  kind.  According 
to  the  side-chain  theory,  if  we  inject  this  bacterium 
into  an  animal  an  over-production  of  the  corres- 
ponding group  will  occur,  resulting  in  a  serum 
which  is  rich  in  body  b.  This  body  b,  however,  is 
not  able  by  itself  to  injure  the  bacteria,  and  a 
bacterium  all  of  whose  receptors  are  laden  with  b 
need  not  at  all  be  injured  in  its  vitality.  Body  b 
normally  possesses  a  peculiar  function,  namely, 
to  serve  as  a  coupling  member  or  link,  and  hence  it 
possesses  two  groups  (amboceptor).  As  has  already 
been  discussed,  one  of  these  groups  fit  the  receptors 
of  the  bacterium  on  the  one  hand  and  the  com- 
plement on  the  other.  When,  therefore,  to  a  normal 
serum  which  contains  suitable  complement,  we  add 
equivalent  amounts  of  immune  serum,  the  con- 
dition pictured  in  A  I  will  result.  On  adding  the 
corresponding  bacterium  to  this  we  get  the  con- 
dition shown  in  A  II,  in  which  all  the  bacterial 
receptors  are  occupied  with  immune  bodies,  or 
more  accurately,  with  immune  bodies  which  on 
their  part  are  loaded  with  bacteriolytic  comple- 
ment c.  in  the  case  here  presented  let  us  say  that 
it  requires  the  occupation  of  all  of  the  receptors 


BACTER10LYSINS  AND  HJEMOLYSINS         IO3 

with  complemented  interbodies  to  cause  the  death 
of  the  bacterium. 

If  now  to  an  equivalent  mixture  of  comple- 
ment and  inter-body  we  add  an  excess  of  inter-body, 
it  will  be  possible  for  only  a  part  of  the  inter-body  to 
be  loaded  with  complement,  leaving  a  portion  of 
the  inter-body  uncomplemented.  On  adding  the 
corresponding  bacteria  a  number  of  conditions  may 
result;  the  affinity  of  the  inter-body  for  the  bac- 
terial receptor  may,  as  a  result  of  the  loading  with 
complement,  (i)  remain  unchanged,  (2)  it  may 
thereby  be  increased,  or  (3)  be  diminished. 

In  the  figure,  B  II  shows  the  condition  of  in- 
creased affinity.  Of  the  six  inter-bodies  only  those 
combine  with  the  bacterium  which  have  become 
laden  with  complement.  In  this  case,  therefore, 
the  excess  of  inter-bodies  will  have  no  influence  on 
the  bactericidal  effect.  The  condition  is  really  the 
same  as  A  II,  except  that  free  inter-body  is  also 
present. 

C  II  shows  the  condition  of  unchanged  affinity. 
In  this  case,  if  we  add  the  bacterium  to  the  mixture 
of  complement  and  excess  of  inter-body,  all  the 
receptors  of  the  bacterium  will,  to  be  sure,  be  occu- 
pied by  inter-bodies,  but  this  will  be  entirely  with- 
out regard  to  the  fact  that  these  inter-bodies  are  or 
are  not  loaded  with  complement.  It  may  there- 
fore happen  that  only  a  few  of  the  bacterial  receptors 
will  be  occupied  by  complemented  (i.e.,  active) 


104  IMMUNE  SERA 

inter-bodies,  while  the  rest  of  the  bacterial  receptors 
are  occupied  by  uncomplemented  (hence  inactive) 
inter-bodies.  As  already  stated,  however,  the 
vitality  of  such  a  bacterium  is  not  necessarily 
destroyed. 

D  II  represents  the  last  conceivable  case.  It  i^ 
assumed  that  the  "  completion  "  of  the  inter-body 
has  resulted  in  a  diminution  of  the  latter 's  affinity 
for  the  bacterial  receptor.  In  this  case  primarily 
only  the  uncomplemented  inter-bodies  will  com- 
bine with  the  bacterial  receptors,  while  the  free 
fluid  will  contain  complemented  inter-bodies. 

In  cases  C  II  and  D  II,  therefore,  the  excess  of 
inter-body  exerts  a  deflecting  action  on  ike  complement, 
thus  diminishing  the  end  results. 

It  is  difficult  to  say  to  what  extent  "  deflection  of 
complement "  really  occurs  in  the  experiments 
referred  to  above.  Studies  by  Buxton,  Gay,  and 
others  show  that  deflection  of  complement  will  not 
always  explain  the  phenomenon,  and  that  in  these 
instances  other  factors  must  be  responsible  for  the 
paradoxical  results. 

Practical  Value  of  Injections  of  Anti-Bacterial 
Sera. — We  use  the  term  "  antibacterial"  advisedly, 
because,  after  all,  when  we  immunize  an  animal 
against  a  certain  bacterium  we  do  not  produce 
merely  a  bactericidal  serum,  but  one  which  con- 
tains agglutinins,  precipitins,  opsonins,  and  per- 
haps still  other  antibodies  as  well.  The  use  of 


BACTERIOLYSINS  AND  H.EMOLYSINS 


specific  antibacterial  sera  has  been  tried  in  man  both 
to  cure  existing  infections  and  as  a  preventive  of 
infection.  The  therapeutic  use  has  in  most  instances 
been  rather  disappointing,  though  in  dysentery, 
plague,  gonococcus  and  meningococcus  infections 
the  results  have  been  somewhat  better.  Recently 
also,  fairly  good  reports  are  heard  from  the  admin- 
istration of  large  doses  of  antistreptococcus  serum. 
In  susceptible  animals  injections  of  some  of  the 
very  virulent  bacteria,  as  pneumococci,  strepto- 
cocci, typhoid  bacilli  and  cholera  spirilla,  can  be 
robbed  of  all  danger  if  small  doses  of  their  respective 
sera  are  given  before  the  bacteria  have  increased  to 
any  great  extent  in  the  body.  If  given  later  they 
are  ineffective.  Conditions  in  man  are  probably 
very  similar. 

The  reasons  for  the  failure  of  these  antibacterial 
sera  when  used  therapeutically  demand  a  moment's 
consideration.  It  is  apparent  from  all  that  has 
gone  before  that  a  deeper  insight  into  the  mechan- 
ism of  immunity  discloses  many  difficulties  to  be 
overcome  before  we  can  hope  for  much  in  a  practical 
way.  In  the  case  of  the  bacteria  sera,  for  example, 
we  have  as  yet  found  no  method  of  increasing  the 
complements,  and  these  are  apparently  highly  im- 
portant in  destroying  the  invading  bacteria.  Nor 
have  we  any  way  to  determine  the  proper  dose  so 
as  to  avoid  the  phenomenon  termed  "deflection  of 
complement."  Possibly,  also,  as  Ehrlich  has  sug- 


106  IMMUNE  SERA 

gested,  the  complements  present  in  human  serum 
may  not  be  able  to  reactivate  immune  bodies 
derived  from  the  horse,  sheep,  or  other  animal 
furnishing  the  therapeutic  serum.  Probably  the 
most  important  cause  of  the  failure  of  these  sera 
is  that  they  do  not  reach  the  bacteria  in  the  body. 
In  the  case  of  cholera,  for  example,  it  is  hardly  to 
be  expected  that  the  serum  injected  would  affect 
the  spirilla,  for  most  of  these  are  in  the  intestinal 
contents,  and  therefore  really  outside  of  the  body. 
In  many  of  the  bacterial  infections  the  organisms 
accumulate  in  the  lymph  glands  and  other  sites 
where  they  cannot  readily  be  reached  by  anti- 
bodies circulating  in  the  blood.  Instructive  in  this 
connection  are  the  good  results  achieved  by  intra- 
spinal  injections  of  antimeningococcus  serum,  when 
exactly  the  same  serum  had  proven  valueless  when 
given  subcutaneously. 


PRECIPITINS 

Definition. — All  ol  the  foregoing  experiments 
have  concerned  themselves  with  the  results  obtained 
by  injection  of  cellular  material  of  one  animal  into 
another.  In  the  further  study  of  this  subject, 
experiments  were  made  to  discover  what  happens 
when  dissolved  albuminous  bodies  of  one  species  are 
injected  into  animals  of  another  species.  This  line 
of  investigation  was  first  pursued  by  Tchistowitsch,1 
who  injected  rabbits  with  the  serum  of  horses  and 
of  eels.  On  withdrawing  serum  from  such  rabbits 
and  mixing  it  with  horse  or  eel  serum,  the  mix- 
ture became  cloudy,  owing  to  the  precipitation  of 
part  of  the  albumin  of  the  horse  or  eel  serum  by 
that  of  the  rabbit.  Normal  rabbit  serum  does  not 
possess  this  property.  Bordet  was  able  to  demon- 
strate that  the  same  thing  takes  place  if  rabbits  are 
treated  with  chicken  blood.  On  mixing  such  a 
serum  with  chicken  serum,  a  precipitate  formed. 
The  substances  which  develop  in  the  serum  by 
treating  an  anima1  with  albuminous  bodies  of 
another  animal,  and  which  precipitate  these  a'bumins 
when  the  sera  of  the  two  animals  are  mixed,  are 

1  Tchistowitsch,  Annal.  Pasteur.  Vol.  xiii,  1899. 
107 


io8  IMMUNE  SERA 

called  precipitins.'1  This  power  of  the  organism  to 
react  to  the  injection  of  foreign  dissolved  albuminous 
substances  has  been  found  to  be  very  extensive. 

Bacterial  Precipitins.  —  In  1897,  R.  Kraus  showed 
that  the  serum  of  a  rabbit  immunized  against 
typhoid  often  produces  a  precipitate  in  the  bac- 
terial-free nitrate  of  a  bouillon  culture  of  typhoid 
bacilli.  This  fact  has  been  verified  by  subsequent 
investigators  and  the  reaction  found  to  be  specific. 
In  general,  the  best  results  are  obtained  with  old 
bouillon  cultures  which  contain  a  larger  proportion 
of  the  autolytic  products.  It  was  natural  that  this 
reaction  should  at  once  be  applied  to  the  diagnosis 
of  typhoid  and  other  diseases.  Numerous  experi- 
ments however  have  shown  that  Kraus'  phenomenon 
is  not  nearly  so  constantly  observed  as  that  of 
agglutination,  and  the  reaction  is  therefore  but 
little  used.  Whether  the  bacterial  precipitins  are 
identical  in  character  with  those  obtained  by 
injecting  an  animal  with  an  unrelated  serum  (zoopre- 
cipitins),  is  still  undecided.  Rostoski,  as  well  as 
Nuttall,  believes  that  they  are  probably  different. 
So  much  for  bacterial  precipitins. 

Lactoserum  —  Other  Specific  Precipitins.  —  Bordet, 
by  injecting  cows'  milk  into  rabbits',  was  able  to 
produce  a  serum  which  precipitates  the  casein  of 

1  It  will  be  recalled  that,  besides  the  production  of  pre- 
cipitins, the  above  procedure  causes  the  formation  of  other 
anti-bodies  such  as  anti-complements,  anti-agglutinins,  etc. 


PREC1PITINS 


log 


cows'  milk.  He  called  this  lactoserum.  Ehrlich, 
Morgenroth,  Wassermann,  Schtitze,  Myers,  and 
Uhlenhuth  showed  that  by  treating  a  rabbit  with 
chicken  albumin  a  precipit'n  is  formed  which  pre- 
cipitates chicken  albumin.  Myers,  by  treating  ani- 
mals with  Witte's  pep  ton  and  globulin,  produced  a 
serum  that  contained  specific  antipeptons  and  anti- 
globulins.  Pick  and  Spiro,  by  using  albumose, 
produced  antialbumoses.  Leclainche  and  Vallee, 
Stern,  Mertens,  and  Ziilzer  treated  animals  with 
human  albuminous  urine  and  produced  a  serum 
which  contained  a  precipitin  specific  for  this  sub- 
stance. Schutze,  by  treating  rabbits  with  a  vege- 
table albumin,  as  well  as  with  human  myoalbumin, 
produced  a  precipitin  specific  for  these  albumins. 
This  does  not  exhaust  the  recital  of  the  work  done 
in  this  field,  and  there  is  a  host  of  other  albuminous 
bodies  which,  when  injected  into  an  animal,  are 
able  to  excite  the  production  of  precipitins. 

Specificity  of  the  Precipitins.  —  It  was  soon  recog- 
nized that  the  specificity  is  not  absolute.  Above  all, 
this  depends  upon  the  strength  of  the  serum,  i.e., 
its  degree  of  activity.  This  is  measured  by  the 
dilution  in  which  it  will  still  react.  Thus  a  highly 
active  serum,  one,  for  example,  which  will  still 
give  a  distinct  reaction  when  diluted  i :  1000  or  over, 
will  produce  a  marked  precipitate  with  the  serum 
used  to  excite  its  production ;  whereas,  in  the  serum 
of  other  animal  species  it  will  produce  slighter  pre- 


HO  IMMUNE  SERA 

cipitates,  or  only  cloudings.  A  less  highly  active 
serum  will  likewise  cause  a  marked  precipitate  in 
the  homologous  blood  solution,  and  a  slight  pre- 
cipitate, or  only  a  clouding,  at  the  most,  in  a  closely 
related  species.  For  example,  the  serum  of  a  rabbit 
which  has  been  treated  with  sheep  blood  produces 
a  marked  precipitate  in  a  solution  of  sheep  blood ;  a 
slight  precipitate  in  a  goat -blood  solution ;  and  a  still 
fainter  one  in  an  ox-blood  solution.  In  some  in- 
stances th2  two  latter  will  show  only  a  clouding.  If 
we  employ  a  very  weak  serum,  even  the  cloudings 
will  be  absent,  and  a  precipitate  is  formed  only  in 
the  sheep-blood  solution.  If  human  blood  or 
blood  serum  has  been  injected,  the  clouding  and 
precipitation  will  occur  most  readily  (aside,  of 
course,  from  human-blood  solution)  in  that  of  apes. 
In  the  precipitin  reaction,  therefore,  the  relationship 
of  the  single  animal  species  is  an  important  factor. 
This  peculiar  behavior  has  first  been  thoroughly 
studied  by  Nuttall  *  who  made  observations  on 
five  hundred  different  animals.  As  a  result  of  these 
we  know  that  a  weak  human-blood  antiserum, 
besides  reacting  on  human  blood,  causes  a  clouding 
only  in  the  blood  of  anthropoid  apes  (chimpanzee, 
gorilla,  orang-outang) ;  a  stronger  serum  causes  a 
clouding  also  in  the  blood  of  other  monkeys ;  finally 

1  British  Medical  Journal,  1901,  Vol.  ii,  and  1902,  Vol.  i. 
See  also  Nuttall,  Blood  Immunity  and  Blood  Relationship, 
1904.  The  Macmillan  Co.,  N.  Y. 


PRECIPITINS  III 

a  very  highly  active  serum  reacts  with  the  blood  of 
all  the  mammalia.  In  that  case,  of  course,  only  a 
faint  clouding  is  produced  even  after  considerable 
time.  Nuttall  also  obtained  antisera,  each  of  which 
was  specific  for  one  of  the  large  animal  classes 
(birds,  reptiles,  amphibia).  Here,  too,  the  same 
quantitative  differences  were  noted. 

Nature  of  the  Precipitins.  —  The  precipitins  are 
fairly  resistant  bodies,  whose  power  gradually 
declines  at  a  temperature  of  60°  C.,  but  is  not  lost 
until  70°  C.  is  reached.  Once  their  action  is  lost, 
it  cannot  be  restored  by  the  addition  of  normal  sera, 
showing  according  to  Ehrlich,  that  the  precipitins 
are  receptors  of  the  second  order  and  are  not  ambo- 
ceptors.  The  resulting  precipitate  is  soluble  in 
weak  acids  and  alkalies.  Peptic  digestion  destroys 
the  substances  which  effect  the  precipitation. 
Leblanc  found  that  the  precipitins  were  precipitated 
from  the  serum  in  that  fraction  which  Hofmeister 
calls  the  pseudo  globulins.  Eisenberg,  on  the  other 
hand,  in  his  experiments  found  them  in  the  eu- 
globulm  fraction.  The  latter  result  was  also  obtained 
by  Obermayer  and  Pick  in  precipitins  obtained 
from  goats  and  rabbits.  The  discordant  results 
are  comprehensible  in  view  of  recent  publications 
concerning  the  unreliability  of  ammonium  sulphate 
fractionation  of  serum  globulins.  The  nature  of 
the  resulting  precipitate  has  also  been  studied  by 
Leblanc.  He  finds  that  it  is  a  combination  of  the 


112  IMMUNE  SERA 

precipitated  albumin  with  the  antibody  of  the 
specific  serum.  In  this  combination  the  properties 
of  the  pseudo  globulin  predominate  showing  that 
it  is  the  specific  serum  which  furnishes  the  greater 
part  of  the  precipitate.  The  presence  of  salts  seems 
to  be  necessary  for  the  precipitin  reaction.  A  tem- 
perature of  37°  C.  hastens,  while  a  low  temperature 
markedly  retards  the  reaction.  In  either  case,  the 
amount  of  precipitum  is  uninfluenced.  The  pres- 
ence of  even  small  quantities  of  acids  or  alkalies 
markedly  reduces  the  amount  of  precipitum  formed, 
but  an  increase  of  salt  (NaCl)  has  little  effect. 

Practical  Application.  —  These  precipitins  have 
very  recently  found  a  practical  application.  Fish, 
Ehrlich,  Morgenroth,  Wassermann,  and  Schutze 
investigated  the  specific  action  of  lactoserum.  They 
found  that  a  serum  derived  by  treating  an  animal 
with  cows'  milk  contained  a  precipitin  which  reacted 
only  on  the  casein  of  cows'  milk,  but  not  on  that  of 
human  milk  or  goats'  milk.  The  serum  of  an  ani- 
mal treated  with  human  milk  was  specific  for  the 
casein  of  human  milk,  etc.  Ehrlich,  Morgenroth, 
and  Wassermann  also  experimented  with  the  serum 
resulting  from  treatment  with  chicken  egg  albumin, 
and  found  that  this,  while  not  strictly  specific  so  far 
as  closely  related  species  are  concerned,  is  yet  so 
against  other  species.  The  precipitins,  therefore, 
react  on  closely  related  albumins,  but  are  specific 
against  those  of  unrelated  species. 


PRECIP1TINS  113 

The  Wassermann-  Uhlenhuth  Blood  Test.  —  As  a 
result  of  these  researches  Wassermann  proposed, 
at  the  Congress  for  Internal  Medicine,  1900,  to  use 
these  sera  as  a  means  of  differentiating  albumins, 
i.e.,  to  distinguish  the  different  albumins  from  one 
another,  and  particularly  to  distinguish  those 
derived  from  man  from  those  of  other  animals. 
This  proposal  thus  to  use  the  Tchistowitsch-Bordet 
precipitins  had  important  practical  and  theoretical 
results.  Uhlenhuth,  Wassermann,  Schutze,  Stern, 
Dieudonne,  and  others  showed  that  a  serum  could 
be  produced  from  rabbits  by  injecting  them  with 
human  serum,  by  means  of  which  it  is  possible  to 
tell  positively  whether  a  given  old,  dried  blood  stain 
is  human  blood  or  not. 

Uhlenhuth  *  tested  nineteen  kinds  of  blood  and 
only  obtained  a  reaction  with  human  blood  upon 
adding  antihuman  serum  to  the  series  of  dilutions. 
He,  moreover,  found  that  human  blood  which  had 
been  dried  four  weeks  on  a  board  could  be  readily 
distinguished  by  means  of  antihuman  serum  from 
the  blood  of  the  horse  and  ox.  On  the  following 
day  Wassermann  2  demonstrated  experiments  simi- 
lar to  Uhlenhuth 's  at  the  meeting  of  the  Physiologi- 
cal Society,  Berlin.  Outside  of  human  blood  only 
that  of  a  monkey  gave  the  reaction  with  anti- 
human  serum. 

1  Uhlenhuth,    Deutsche    med.    Wochenschrift,    1901.     xxvii. 
*  Wassermann  A.  and    Schutze,  Berliner  klin.  Wochenschr. 
1901.     No.  xxviii. 


114  IMMUNE  SERA 

The  reliability  of  this  reaction  in  medico-legal 
questions  has  been  abundantly  established.  In  the 
forensic  blood  diagnosis  the  subjects  of  the  test 
are  usually  blood  stains  on  clothing,  and  on  wood 
and  metal  objects.  After  such  a  doubtful  stain 
has  been  dissolved  in  physiological  salt  solution, 
one  first  proceeds  to  determine  that  it  is  really 
blood.  For  this  purpose  Teichmann's  test  (the 
production  of  haemin  crystals),  the  guaiac  test,  and 
the  spectroscopic  examination  are  undertaken.  This 
is  of  considerable  importance,  for  not  merely  blood 
but  other  albuminous  solutions  derived  from  the 
same  animal  react  with  an  antiserum  obtained  by 
injecting  an  animal  with  blood  or  serum,  ^Having 
found  that  the  stain  is  that  of  blood,  we  next  deter- 
mine the  special  kind  of  blood. 

Immunizing  the  Animals.  —  For  the  production  of 
the  antisera,  we  make  use  of  rabbits.  These  can  be 
injected  either  with  sterile,  freshly-defibrinated  blood 
or  with  sterile  serum,  the  latter  being  preferable 
for  intravenous  inoculation.  It  is  well  to  begin 
with  small  doses  and  gradually  increase;  thus  for 
intravenous  inoculations  the  first  injection  should 
be  about  one  c.c.  and  increased  up  to  three  or 
four  c.c.  With  intraperitoneal  injections  about 
double  these  doses  can  be  given.  Ordinarily,  the 
interval  between  injections  is  three  or  four  days, 
and  the  entire  duration  of  treatment  from  two 
weeks  to  a  month.  Long-continued  treatment 


PRECIPITINS  115 

leads   to   a  disappearance   of   precipitins  from   the 
blood. 

Collecting  the  Serum.  — When  the  animals  have 
received  five  to  six  injections,  and  some  days  have 
elapsed  it  is  well  to  draw  off  samples  of  the  blood 
and  to  test  for  precipitins.  This  is  easily  done  by 
shaving  the  ear  and  cleansing  the  skin  with  alcohol 
and  sterile  water.  An  incision  is  then  made  into 
the  marginal  vein  and  a  few  drops  of  blood  collected 
in  a  small  test-tube.  This  is  then  set  aside  to  allow 
the  blood  to  coagulate.  After  the  serum  has  sepa- 
rated it  can  be  tested  and  if  it  prove  insufficiently 
powerful,  treatment  may  be  continued,  otherwise 
the  animal  may  be  killed,  preferably  a  week  or  ten 
days  after  the  last  injection.  The  animals  may  be 
killed  in  a  variety  of  ways.  Uhlenhuth  chloroforms 
them,  opens  the  thoracic  cavity  under  aseptic 
precautions,  and,  cutting  through  the  beating  heart, 
the  blood  is  allowed  to  flow  into  the  thoracic  cavity, 
whence  it  is  removed  by  means  of  sterile  pipettes 
to  suitable  vessels.  Nuttall's  method  is  to  shave 
the  neck  and  disinfect  the  skin  with  lysol  solution; 
bend  the  animal's  head  backward  to  put  the  skin 
of  the  neck  on  the  stretch,  and  have  an  assistant 
make  a  clean  sweep  with  a  sterilized  knife  through 
the  tense  skin  to  and  through  the  vessels.  The 
blood  spurts  into  a  large  sterile  dish  which  is 
immediately  covered  when  the  main  flow  has  ceased. 
The  dishes  are  placed  horizontally  until  a  clot  has 


Il6  IMMUNE  SERA 

formed ;  they  are  then  slightly  tilted,  and  as  soon  as 
serum  enough  has  been  expressed,  this  is  pipetted 
off  into  sterile  test  containers  which  are  stored  in  a 
cool  place.  It  is  well  not  to  add  any  preservative 
to  the  serum,  as  such  an  addition  may  occasionally 
lead  to  pseudo  reactions. 

The  Test.  —  In  carrying  out  the  test  the  sus- 
pected clot  is  mixed  with  a  small  quantity  of  normal 
salt  solution  and  then  filtered.  Whether  or  not  the 
blood  specimen  has  gone  into  solution  can  best  be 
judged  by  the  foam  test.  Air  is  blown  gently  through 
the  pipette  which  is  used  for  transferring  the  solu- 
tion into  the  test-tubes.  Solutions  of  blood  or 
serum  of  i :  1000  and  over,  still  foam  well.  The  color 
of  the  fluid  is  not  so  reliable  an  index  of  solution. 
To  some  of  this  solution  in  a  test-tube,  about  double 
the  amount  of  the  specific  serum  (derived  as  above) 
is  added.  As  a  control  test,  we  place  a  little  blood 
of  another  species,  e.g.,  of  an  ox,  in  a  second  test- 
tube  together  with  some  of  the  specific  serum  and  a 
little  normal  salt  solution.  In  a  third  tube  we 
place  some  of  the  suspected  blood  solution,  and  'n  a 
fourth  some  of  the  specific  serum  mixed  with  the 
normal  salt  solution.  All  four  tubes  are  placed  in 
the  incubator  at  37°C.  for  one  hour,  or  are  left  at 
room  temperature  for  several  hours.  If  the  sus- 
pected clot  was  one  of  human  blood,  the  first  tube 
will  show  distinct  evidence  of  precipitation,  while 
all  the  control  tubes  will  have  remained  clear.  It 


PRECIPITINS 


117 


is  desirable  to  dilute  the  suspected  blood  as  far  as 
possible  when  testing,  for  when  concentrated  sera 
are  brought  together  reactions  may  occur  which 
will  lead  to  erroneous  conclusions.  In  medico- 
legal  work  it  will  be  well  to  progressively  dilute  a 
suspected  blood  sample  and  to  reach  a  conclusion 
upon  the  highest  (within  limits)  which  reacts  to  a 
given  antiserum.  In  routine  work  one  can  com- 
mence with  dilutions  of  the  suspected  blood  of 
i :  100  or  i :  200.  We  must  not  omit  to  say  that  it 
is  necessary  to  test  to  litmus  all  solutions  to  be 
examined,  and  to  neutralize  any  that  are  found 
decidedly  acid  or  alkaline. 

Appearance  of  the  Reaction.  —  When  antiserum 
is  added  to  blood  dilution  it  sinks  to  the  bottom  of 
the  tube,  forming  a  milky  white  zone  at  the  point  of 
contact.  The  milkiness  gradually  extends  upward 
until  the  whole  fluid  is  clouded.  Where  the  fluids 
have  been  mixed  by  shaking  this  diffuse  cloudiness 
undergoes  a  change;  after  ten  to  twenty  minutes, 
or  later,  very  fine  granules  of  precipitum  begin  to 
appear,  and  the  upper  layers  of  the  fluid  begin  to 
clear,  due  to  sedimentation  of  the  precipitum. 
The  fine  particles  soon  become  aggregated  into 
coarser  ones,  and  these  into  flocculi  which,  gradually 
sinking  to  the  bottom  of  the  tube,  give  rise  to  more 
or  less  deposit  of  a  whitish  appearance.  With  blood 
dilutions  of,  say  i :  40  to  i :  200  and  over,  the  deposit 
formed  is  usually  sharply  defined ;  where  more  con- 


Ii8  IMMUNE  SERA 

centrated  dilutions  are  used,  the  deposit  may  form 
an  irregular  mass  at  the  bottom  of  the  tube. 

The  reaction  may  be  followed  microscopically  by 
means  of  the  hanging-drop  method.  By  this  method 
a  reaction  can  be  observed  within  ten  to  fifteen 
minutes,  which  macroscopically  becomes  visible 
only  after  two  hours. 

Delicacy  of  the  Precipitin  Test.  —  Whereas  the 
ordinary  chemical  tests  for  blood  cease  to  give  reac- 
tions in  dilutions  of  about  i :  1000,  powerful  antisera 
greatly  exceed  this  limit,  as  the  reported  results  of 
independent  observers  have  shown.  Working  with 
an  antihuman  serum,  Strube  reports  a  re.action 
with  a  blood  diluted  20,000  times,  and  Stern  one 
with  a  blood  diluted  50,000  times.  Ascoli  obtained 
a  reaction  with  a  specific  serum  with  egg  albumin 
diluted  1,000,000  times. 

Other  Applications  of  the  Precipitin  Test.  —  It  can 
be  readily  understood  that  this  test  finds  ready 
application  in  the  detection  of  horse,  dog,  or  cat 
meat  in  sausage. 

The  principle  and  the  method  are  the  same  in  all 
these  various  applications.  We  treat  animals  with 
the  albumins  which  we  wish  to  differentiate,  and  so 
obtain  sera  specific,  each  for  its  particular  kind  of 
albumin.  These  sera,  then,  produce  precipitates 
only  in  solutions  of  their  respective  albumins.  For 
example,  if  we  wish  to  determine  whether  a  given 


PREC1P1T1NS 


Iig 


sample  of  meat  is  horse-flesh  or  not  we  must  inject 
an  animal  with  horse  serum,  or,  if  we  prefer,  with 
an  extract  of  horse-flesh.  The  serum  derived  from 
this  animal  will  then  produce  a  precipitate  in  the 
aqueous  extract  of  the  meat  if  this  be  horse-flesh, 
but  not  if  it  be  beef.  Animals  treated  with  dog 
serum  yield  a  serum  which  precipitates  an  aqueous 
extract  of  dog-flesh,  etc.  The  method  of  examina- 
tion consists  in  scraping  the  meat  and  extracting  it 
with  water  or  normal  salt  solution.  It  takes  a  long 
time  to  extract  the  meat  in  some  cases.  An  extract 
is  suitable  for  testing  when  it  foams  on  being  shaken. 
If  the  extract  is  very  cloudy  it  should  be  cleared  by 
filtration  through  a  Berkfeld  filter.  In  testing,  add 
ten  to  fifteen  drops  of  antiserum  to  3  cc.  of  the 
saline  meat  extract. 

Antiprecipitins  —  Iso-precipitins.  —  Biologically, 
the  precipitins  are  found  to  behave  -like  the  sub- 
stances already  studied.  It  is  possible,  for  example, 
by  injecting  an  animal  with  a  precipltin,  say 
lactoserum,  to  obtain  an  antiprecipiiin,  an  anti- 
lactoserum,  which  counteracts  or  inhibits  the 
action  of  the  precipitin.  This  is  entirely  analo- 
gous to  the  antihaemolysins,  the  an tispermo toxin, 
etc. 

If  rabbits  are  treated  with  rabbit  serum,  a  serum 
is  obtained  which  will,  in  certain  cases,  precipitate 
the  serum  of  other  rabbits.  This  was  done  by 


120  IMMUNE  SERA 

Schutze,  and  he  called  this  serum  iso-precipitin. 
Whether  or  not  iso-precipitins  ever  occur  in  normal 
serum  has  not  yet  been  definitely  established. 
Their  occurrence  in  human  serum  might  be  of 
importance  in  homologous  transfusion. 


II.   CYTOTOXINS 

Cytotoxins  —  Definition  —  Leucotoxin  —  Nature  of 
the  Cytotoxin  —  Anticytotoxin.  —  After  it  had  been 
found  that  the  injection  of  an  animal  with  red  blood 
cells  of  another  animal  was  followed  by  the  produc- 
tion of  definite,  specific  reaction  substances,  investi- 
gators experimented  to  see  whether  this  was  also 
the  case  if  other  animal  cells  were  used.  Injections 
were  made  with  white  blood  cells,  spermatozoa  of 
other  animals,  etc.,  and  there  resulted  a  series  of 
reaction  substances,  entirely  analogous  to  the 
haemolysins,  which  were  specific  for  the  cells  used  for 
injection.  These  sera  Metchnikoff  calls  cytotoxins. 
After  Delezenne  had  published  a  short  article  on  a 
serum  haemolytic  for  white  blood  cells,  Metchnikoff 
undertook  a  study  of  the  substances  produced  in 
sera  of  animals  treated  with  leucocytes  of  another 
species.  He  injected  guinea  pigs  with  the  mesen- 
teric  glands  and  bone  marrow  of  a  rabbit.  He 
also  injected  for  several  weeks  half  an  Aselli's  pan- 
creas at  a  time,  at  intervals  of  four  days.  If  he 
withdrew  serum  from  such  a  guinea  pig  he  found 
this  to  be  intensely  solvent  for  white  blood  cells  of 
a  rabbit.  He  called  this  serum  leucotoxin.  This 
leucotoxin  is  very  poisonous  for  these  animals,  and 

121 


122  IMMUNE  SERA 

kills  them  within  a  few  hours.  Non-fatal  doses  at 
first  excite  a  marked  hypoleucocytosis,  which  is 
followed  after  a  few  days  by  a  compensatory  hyper- 
leucocytosis.  Leucotoxin  destroys  the  mononu- 
clear  as  well  as  the  polynuclear  leucocytes  of  the 
animal,  as  was  shown  by  Funk.  Leucotoxin  which 
had  been  derived  by  injection  of  the  leucocytes  of 
horses,  oxen,  sheep,  goats,  or  dogs  acted  only  on 
the  leucocytes  of  that  species,  not  on  the  leucocytes 
of  man.  So  far  as  the  mechanism  of  the  cy  to  toxic 
action  is  concerned,  it  has  been  found  that  this  is 
the  same  as  that  of  the  hasmolysins.  The  action  of 
the  specific  cytotoxic  serum  is  always  due  to  the 
combined  action  of  two  substances  in  the  serum,  a 
specific  immune  body,  and  an  alexin  or  comple- 
ment present  also  in  normal  serum.  The  cyto- 
toxic sera,  like  the  haemolytic  sera,  are  rendered 
inactive  by  heating  to  55°  C.  In  other  respects 
also  the  cytotoxic  sera  maintain  the  analogy  to 
the  haemolytic  sera.  Thus  it  is  possible  by  immu- 
nizing with  a  cy  to  toxin  to  obtain  an  anticytotoxin. 
MetchnikofT,  for  example,  was  able  to  produce'  an 
antileucotoxin  by  injecting  animals  with  leuco- 
toxin.  This  antibody  inhibited  the  action  of  the 
leucotoxin. 

Neurotoxin.  —  Delezenne  and  Madame  Metchni- 
koff  have  injected  animals  with  central-nervous- 
system  substance,  and  so  produced  a  specific  neuro- 
ioxin.  They  injected  ducks  intraperitoneally,  giving 


CYTOTOX1NS  123 

them  five  or  six  injections  of  ten  to  twenty  grammes 
of  dog  brain  and  spinal  cord  mixed  with  normal 
salt  solution.  The  serum  of  these  ducks  injected 
intracerebrally  into  dogs  in  doses  of  0.5  c.c. 
caused  the  dogs  to  die  almost  at  once  in  complete 
paralysis,  whereas  if  normal  duck  serum  was  in- 
jected in  the  same  way  no  effects  of  any  kind  were 
produced.  If  smaller  doses  of  the  specific  neuro- 
toxic  serum  were  administered,  say  o.i  to  0.2  c.c., 
various  paralyses  and  epileptiform  convulsions  set 
in,  from  which  the  animals  sometimes  recovered. 
The  action  of  this  serum  is  specific,  i.e.,  the  serum 
of  ducks  treated  with  dog  brain  causes  these  symp- 
toms only  in  dogs,  while  on  rabbits  it  acts  no 
differently  than  normal  duck  serum. 

Spermatoxin.  —  Another  specific  cell-dissolving 
serum  was  produced  by  Landsteiner,  Metchnikoff, 
and  Moxter,  by  injecting  animals  with  the  sperma- 
tozoa of  other  animals.  Such  a  serum  rapidly 
destroys  the  spermatozoa  of  the  animals  whose 
product  was  injected.  This  cytotoxin  was  named 
spermatoxin.  If  animals  are  treated  with  spermato- 
zoa there  is  produced  a  serum  which  is  not  only  a 
spermatoxin,  but  which  is  also  hasmolytic  for  the 
red  cells  of  that  animal.  This  was  demonstrated 
by  Metchnikoff  and  Moxter,  and  has  already  been 
referred  to  in  discussing  hsemolysins.  If,  for  ex- 
ample, we  inject  the  spermatozoa  of  sheep  into 
rabbits,  we  shall  obtain  a  serum  that  is  sperma- 


124  IMMUNE  SERA 

toxic  for  sheep,  as  well  as  haemolytic  for  sheep 
red  cells. 

Common  Receptors.  —  At  first  it  was  thought  that 
the  haemolysin  so  produced  was  due  to  the  presence 
of  small  quantities  of  blood  injected  with  the  sper- 
matozoa. The  same  result  however  was  obtained 
when  all  traces  of  blood  could  be  excluded  ;*  further- 
more a  number  of  investigators  produced  haemoly- 
sins  by  the  injection  of  fluids  entirely  free  from  red 
corpuscles,  such  as  serum  and  urine.  The  produc- 
tion of  this  haemolysin  is  not  hard  to  explain  if  we 
hold  fast  to  the  side-chain  theory.  We  have 
merely  to  assume  that  the  spermatozoa  or  these 
other  substances  possess  certain  receptors  in  com- 
mon with  the  red  blood  cells  of  the  same  animal. 
Ehrlich  and  Morgenroth 2  have  repeatedly  pointed 
out  that  specificity  is  a  matter  not  of  cells,  but  of 
receptors.  Despite  these  very  conclusive  demon- 
strations later  investigators,  who  attempted  to 
produce  antisera  for  the  cells  of  various  organs, 
continued  to  use  emulsions  of  unwashed  organs,  in 
utter  disregard  of  the  presence  of  free  receptors  in 
the  organ  juices  and  also  without  consideration  of 
the  antibodies  certain  to  be  produced  by  the  red 
cells  normally  present. 

Cytotoxin  for  Epithelium.  —  As  far  back  as   1899, 

1  Von  Dungern.    See  "Collected  Studies  on  Immunity, "  Ehr- 
lich-Bolduan,  p.  47.     Wiley  &  Sons,  New  York,  1910. 

2  Ehrlich  and  Morgenroth.     Ibid.,  p.  100. 


CYTOTOX1NS  125 

von  Dungern  showed  that  it  was  possible  to  produce 
an  antiepithelial  serum  by  treating  animals  with 
the  ciliated  tracheal  epithelium  of  oxen.  This 
serum  was  rapidly  destructive  for  this  particular 
kind  of  epithelium,  but  it  contained  also  a  specific 
haemolytic  body  just  as  was  the  case  in  the  sper- 
motoxic  serum,  and  for  the  same  reasons.  This 
antiepithelial  serum  aroused  considerable  interest 
since  it  indicated  the  possibility  of  producing  sera 
which  were  cytotoxic  for  certain  varieties  of  epi- 
thelial cells,  especially  those  of  pathological  origin, 
as  carcinoma.  The  numerous  experiments  made 
in  this  direction  failed  however  to  produce  the 
desired  results.  Owing  to  the  extensive  distribu- 
tion of  common  receptors  the  antisera  were  found 
to  exhibit  quite  general  properties  and  to  lack 
that  degree  of  cell  specificity,  essential  for  practical 
purposes. 

Cytotoxins  by  the  Use  of  Nucleo-Proteids.  —  In 
order  to  prevent  the  adventitious  formation  of 
those  bodies  resulting  from  impure  methods  of 
immunization,  and  also  in  the  hope  of  obtaining 
greater  specificity,  a  few  investigators  have  utilized 
the  nucleo-proteids  of  the  cell  for  immunization. 
This  method  seems  to  have  been  tried  first  by 
Marrassini  in  1903,  but  with  indifferent  results. 
In  1905  Beebe1  published  an  extensive  study  along 

1     S.   P.  Beebe,  Cytotoxic  Serum  Produced  by  the  Injection 
of  Nucleo-Proteids.     Journ.  Exper.  Medicine,  Vol  vii,  1905. 


126  IMMUNE  SERA 

these  lines  and  described  the  formation  of  a  nephro- 
toxic  serum  which  caused  albuminuria  and  acute 
degeneration  of  the  kidney  without  changes  in 
the  other  organs.  Albuminuria  appeared  gene- 
rally on  the  fourth  or  fifth  day,  increased  rapidly 
in  amount,  and  was  accompanied  by  the  excretion 
of  hyaline  and  granular  casts.  Subsequently  Pearce 
and  Jackson,1  after  a  careful  experimental  study  on 
the  production  of  cytotoxic  sera  by  the  injection  of 
nucleo-proteids,  conclude  "  that  the  results  do  not 
support  the  theory  that  specific  cytotox!c  sera  may 
be  developed  in  this  way,  but  indicate,  rather,  that 
such  sera  have  certain  mildly  toxic  properties  acting 
in  a  general  way  and  affecting  especially  the  principal 
excretory  organ,  the  kidney." 

1  R.  M.  Pearce  and  Holmes  Jackson,  Journal  of    Infectious 
Diseases,  Vol.  iii,  1906. 


OPSONINS  OR   BACTERIOTROPIC 
SUBSTANCES 

Historical.  -  -  The  early  work  of  Nuttall  and 
others  on  the  bactericidal  action  of  normal  serum, 
and  Pfeiffer's  demonstration  of  the  bacteriolysis 
of  cholera  and  typhoid  bacilli  by  immune  sera  in  the 
absence  of  cells,  formed  the  chief  basis  on  which 
rested  the  humoral  theory,  which  attributed  the 
protection  in  such  cases  to  the  destructive  action 
of  the  serum  on  the  microbes.  It  was  found,  how- 
ever, that  cases  of  protection  resulting  from  the 
use  of  immune  serum  occurred  where  no  such 
bacteriolytic  action  could  be  demonstrated;  infec- 
tion with  plague  or  streptococcus  may  be  men- 
tioned as  examples.  It  is  now  pretty  generally 
accepted  that  immunity  in  these  cases  is  due  largely 
to  the  phagocytic  action  of  the  leucocytes.  As  far 
back  as  1858  Haeckel  had  observed  that  particles 
of  indigo  injected  into  the  veins  of  certain  molluscs 
could  shortly  afterwards  be  found  in  the  blood 
cells  of  the  animal.  However,  the  significance  of 
this  and  other  observations  was  not  appreciated 
until  Metchnikoff  *  in  1883  called  attention  to  their 
bearing  on  infection  and  immunity.  The  outcome 

1  Arbeiten  des  Zoo  log.  Institutes  in  Wien,  1883,  Vol.  v. 

127 


128  IMMUNE   SERA 

of  his  investigations  was  the  establishment  of  the 
well-known  doctrine  of  phagocytosis,  the  principle 
of  which  is  that  the  wandering  cells  of  the  animal 
organism,  the  leucocytes,  possess  the  property  of 
taking  up,  rendering  inert,  and  digesting  micro- 
organisms which  they  may  encounter  in  the  tissues. 
Metchnikoff  believes  that  susceptibility  to  or 
immunity  from  infection  is  essentially  a  matter 
between  the  invading  bacteria  on  the  one  hand 
and  the  leucocytes  of  the  tissues  on  the  other.  He 
realizes  that  the  serum  constituents  play  an  im- 
portant role,  but  this  role  consists  in  their  stimulat- 
ing the  leucocyte  to  take  up  the  bacteria. 

Thus  if  a  highly  virulent  organism  is  injected 
into  a  susceptible  animal,  the  leucocytes  appear  to 
be  repelled,  and  to  be  unable  to  deal  with  the 
microbe,  which  multiplies  and  causes  the  death  of 
the  animal.  If,  however,  the  suitable  immune 
serum  is  injected  into  the  animal  before  inoculation, 
the  phagocytes  attack  and  devour  the  invading 
micro-organisms.  Admitting  that  the  phagocyte 
plays  an  important  part  in  certain  infections  the 
question  must  still  be  considered  whether  the 
immune  serum  has  acted  on  the  injected  microbes 
or  on  the  phagocytes.  Metchnikoff,  we  have  seen, 
takes  the  latter  view. 

In  1903  A.  E.  Wright 1  called  attention  to  certain 
substances  present  in  serum  which  acted  on  bacteria 

1  Wright  and  Douglas,  Proc.  Royal  Society,  Vol.  72,  1903. 


OPSONINS  129 

and  rendered  them  mere  easily  taken  up  by  the 
phagocytic  cells.  He  called  this  substance  opsonin 
and  showed  that  it  is  present  in  normal  as  well  as 
immune  sera.  By  means  of  absorption  tests 
modelled  after  those  of  Ehrlich  and  Morgenroth,  he 
showed  that  the  opsonin  has  a  specific  affinity  for 
the  bacteria  and  none  for  the  leucocytes.  The 
opsonins  for  staphylococcus  prepare  only  staphy- 
lococci  for  the  leucocytes,  those  for  tubercle  bacilli 
only  these  bacteria,  etc.  As  a  result  of  his  obser- 
vations Wright  supposes  that  the  phagocytes  play 
only  a  passive  r6le,  which  depends  on  the  pre- 
liminary action  of  the  opsonin. 

Bacteriotropic  Substances.  -  -  Independently  of 
Wright,  though  somewhat  later,  Neufeld  and  Rim- 
pau  l  of  Berlin  published  experiments  on  the  pha- 
gocytic effect  of  immune  sera.  They  also  found 
that  in  these  sera  there  exists  a  substance  which  has 
no  direct  action  on  the  phagocytes,  but  which  can 
fix  itself  on  the  corresponding  bacteria  and  so  modify 
these  that  they  are  more  readily  devoured  by  the 
phagocytes.  They  call  this  constituent  a  "  bacte- 
riotropic  substance."  There  is  little  doubt  that  this 
bacteriotropic  substance  and  Wright's  opsonin  are 
identical.  Certain  differences  in  the  effect  of  heat 
are  probably  to  be  explained  by  the  differences  in 
the  quantities  of  these  sensitizing  substances  in 
normal  and  immune  sera. 

1  Neufeld  and  Rimpau,  Deutsche  med.  Wochenschrift,  1904. 


IMMUNE  SERA 

Opsonins  Distinct  Antibodies.  —  It  was  natural  to 
question  whether  these  "  opsonins  "  were  really  dis- 
tinct from  other  antibodies,  or  whether  they  were 
perhaps  identical  with  the  immune  body  (or  sub- 
stance sensibilatrice).  In  a  series  of  papers  on  this 
subject  Hektoen  l  shows  that  the  former  is  the  case, 
opsonins  are  distinct  substances.  This  is  not  only 
indicated  by  the  results  of  absorption  tests,  but  by 
the  fact  that,  by  immunization,  a  serum  can  in  cer- 
tain cases  be  obtained  which  is  opsonic  but  not  lytic, 
or  in  other  cases  one  which  is  lytic  but  not  opsonic. 
Similar  experiments  have  differentiated  opsonins 
from  agglutinins. 

Structure  of  Opsonins. — In  structure  the  opso- 
nins are  like  the  agglutinins.  Following  Ehrlich's 
conceptions  they  possess  two  groups,  opsoniferous 
end  haptophore.  On  heating  an  opsonic  serum 
the  former  group  is  destroyed,  but  the  haptophore 
group  remains  intact,  as  can  be  seen  from  suitable 
combining  experiments.  There  is  still  consider- 
able difference  of  opinion  as  to  the  degree  of  heat 
necessary  to  inactivate  the  opsonins.  Once  the 
opsoniferous  group  has  been  destroyed  it  is  impos- 
sible to  restore  the  opsonic  action  by  the  addition 
of  a  complementing  substance.  Hence  the  opsonins 
are  to  be  regarded  as  receptors  of  the  second  order 
and  similar  in  structure  to  the  agglutinins  and 
precipitins.  In  this  connection  it  will  be  we.ll  to 

1  Hektoen,  L.,  Journal  Infect.  Diseases,  1905  and  1906. 


OPSONJNS  I3i 

remember  Bordet's  objections  to  the  assumption 
of  two  groups  in  the  agglutinin  molecule.  These 
have  already  been  considered  oh  page  40. 

The  Opsonic  Index. — In  the  study  of  these  opso- 
nins  Wright  developed  the  idea  that  they  were 
highly  important  in  combating  a  number  of  bacterial 
infections,  such  as  staphylococcus  and  tubercle. 
His  observations  showed  that  inoculations  of  the 
corresponding  bacteria  produced  marked  changes  in 
the  opsonic  contents  of  the  infected  individual  and 
that  it  was  possible  to  estimate  accurately  the  im- 
munizing effect  of  such  inoculations. 

Technique.  —  Wright's  technique  of  measuring  the 
opsonic  power  is  a  slight  modification  of  the  Leish- 
man l  method  and  is  as  follows :  An  emulsion  of 
fresh  human  leucocytes  is  made  by  dropping  twenty 
drops  of  blood  from  a  finger  prick  into  20  c.c. 
normal  salt  solution  containing  one  per  cent  sodium 
citrate.  The  mixture  is  centrifuged,  the  supernatant 
clear  fluid,  removed  and  the  upper  layers  of  the  sedi- 
mented  blood  cells  transferred  by  means  of  a  fine 
pipette  to  10  c.c.  normal  salt  solution.  After  cen- 
trifuging  this  second  mixture  the  supernatant  fluid 
is  pipetted  off  and  the  remaining  suspension  used 
for  the  opsonic  tests.  Such  a  "  leucocyte  emulsion,  ' 
of  course,  contains  an  enormous  number  of  red 
blood  cells;  the  proportion  of  leucocytes,  however, 
is  greater  than  in  the  original  blood. 

1  Leishman,  British  Medical  Journal,  Jan.,  1902. 


132  IMMUNE  SERA 

One  volume  of  this  emulsion  is  mixed  with  one 
volume  of  the  bacterial  suspension  to  be  tested  and 
with  one  volume  of  the  serum.  This  is  best  accom- 
plished by  means  of  a  pipette  whose  end  has  been 
drawn  out  into  a  capillary  tube  several  inches  in 
length.  With  a  mark  made  about  three-quarters 
of  an  inch  from  the  end  it  is  easy  to  suck  up  one  such 
volume  of  each  of  the  fluids,  allowing  a  imall  air 
bubble  to  intervene  between  each  volume.  All 
three  are  now  expelled  on  a  slide  and  thoroughly 
mixed  by  drawing  back  and  forth  into  the  pipette. 
Then  the  mixture  is  sucked  into  the  pipette,  the  end 
sealed  and  the  whole  put  into  the  incubator  at  37°  C. 
The  identical  test  is  made  using  a  normal  serum  in 
place  of  the  serum  to  be  tested.  Both  tubes  are 
allowed  to  incubate  fifteen  minutes  and  then  ex- 
amined by  means  of  smear  preparations  on  slides 
spread  and  stained  in  the  usual  way.  The  degree  of 
phagocytosis  is  then  determined  in  each  by  count- 
ing a  consecutive  series  of  fifty  leucocytes  and  find- 
ing the  average  number  of  bacteria  ingested  per 
leucocyte.  This  number  for  the  serum  to  be  tested 
is  divided  by  the  number  obtained  with  the  normal 
serum  and  the  result  regarded  as  the  opsonic  index 
of  the  serum  in  question.  The  presence  of  a  high 
opsonic  index  Wright  regards  as  indicative  of  in- 
creased resistance.  He  further  states  that  the  fluc- 
tuation of  the  opsonic  index  in  normal  healthy 
individuals  is  not  more  than  from  .8  to  1.2,  and  that 


OPSONINS 


133 


an  index  below  .8  is  therefore  almost  diagnostic  of 
the  presence  of  an  infection  with  the  organism  tested. 
Application  of  the  Opsonic  Measurements.  —  At 
the  present  time  Wright  has  correlated  all  his  obser- 
vations and  built  up  a  system  of  treating  bacterial 
infections  by  means  of  active  immunization  con- 
trolled by  opsonic  measurements.  The  principles 
underlying  his  method  may  be  briefly  summarized 
as  follows:  In  localized  bacterial  infections  the 
infected  body  absorbs  but  small  amounts  of  bacterial 
substances  or  antigens.  In  consequence  of  this  the 
amount  of  active  immunity  developed  is  but  slight. 
Localized  infections  therefore  tend  to  run  a  chronic 
course.  The  logical  method  of  effecting  a  cure  in 
these  cases  is  to  actively  immunize  the  body  with 
the  invading  organism.  In  a  number  of  infections, 
notably  those  of  staphylococcus,  streptococcus,  and 
tubercle,  the  degree  of  immunity  is  measured  accu- 
rately by  the  opsonic  index.  Following  an  inocu- 
lation with  the  infecting  bacteria  (dead  cultures  in 
salt  solution)  there  is  first  a  drop  in  the  opsonic 
index,  the  "  negative  phase,"  then,  depending  on 
the  size  of  the  dose  and  the  reacting  power  of  the 
individual,  there  comes  a  rise  of  the  index,  the 
"  positive  phase,"  or  a  continuation  of  the  negative 
phase.  The  former  is  obtained  with  proper  dosage ; 
the  latter  with  doses  too  large  or  too  small.  In 
estimating  the  size  of  the  dose  given,  Wright  counts 
the  number  of  bacteria  per  c.c.  of  emulsion  injected. 


134  IMMUXE  SERA 

Thus  in  the  case  of  localized  staphylococcus  infec- 
tions the  doses  for  adult  humans  range  from  100 
million  to  500  million  bacteria.  In  the  case  of  strep- 
tococcus the  doses  are  smaller,  averaging  about  50 
to  100  million.  The  bacterial  suspensions  are  heated 
to  60°  C.  for  twenty  minutes,  0.5%  carbolic  acid 
is  added,  and  tests  are  made  to  insure  sterility. 
The  time  for  inoculation  is  governed  by  the  opsonic 
index.  If  the  first  inoculation  has  been  properly 
gauged  there  is  a  brief  negative  phase,  followed  by 
a  positive  phase  of  some  days'  duration.  As  this 
positive  phase  gradually  drops,  one  gives  another 
inoculation  and  watches  the  effect  on  .the  opsonic 
index.  If  the  index  drops  markedly  and  rises  but 
little,  the  dose  has  been  too  large.  Or  if  the  nega- 
tive phase  is  slight,  and  the  positive  phase  slight  and 
transitory,  the  dose  has  been  too  small.  With 
proper  dosage  the  negative  phases  are  small,  and  the 
opsonic  index  is  kept  fairly  well  above  normal. 
Hand  in  hand  with  this  goes  an  improvement  in  the 
clinical  symptoms. 

Wright  and  his  pupils  have  published  accounts  of 
a  large  number  of  cases  successfully  treated  accord- 
ing to  this  method.  The  results  are  reported  as  espe- 
cially good  in  cases  of  severe  acne,  multiple  boils, 
lupus,  tubercular  glands,  and  bone  tuberculosis. 

In  judging  of  the  value  of  Wright's  method  we 
must  bear  clearly  in  mind  that  the  essential  feature 
of  it  is  the  control  by  opsonic  measurements;  treat- 


OPSONINS  135 

ment  of  bacterial  infections  by  the  inoculation  of 
dead  cultures  has  long  been  known. 

The  results  obtained  by  most  workers  in  this  coun- 
try fail  to  bear  out  Wright's  claims  for  the  method. 
Thus  the  author1  finds  that  the  variation  in  the 
opsonic  indices  of  several  normal  persons  is  often 
considerable;  that  opsonic  counts  based  on  fifty 
leucocytes  may  occasionally  vary  by  more  than 
50%  and  that  it  is  therefore  necessary  to  count  from 
150  to  200  leucocytes  for  each  test;  that  duplicate, 
triplicate  and  more  tests  made  of  the  same  serum, 
at  the  same  time,  and  under  identical  conditions  so 
far  as  one  can  tell,  frequently  give  widely  divergent 
results;  that  the  opsonic  index  and  the  clinical 
course  of  the  disease  do  not  always  run  parallel. 
Cases  may  do  very  well  and  have  the  index  remain 
low;  other  cases  may  do  poorly  with  an  increased 
opsonic  index.  It  is  to  be  noted,  furthermore,  that 
some  of  these  variations  in  results  are  unavoidable, 
at  least  with  the  present  technique. 

To  one  who  has  followed  the  progress  of  immunity 
studies,  it  is  not  at  all  surprising  to  find  that  the 
opsonic  index  is  not  necessarily  a  measure  of  the 
patient's  immunity.  When  Gruber  and  Durham 
published  their  observations  on  agglutinins  the 
phenomenon  was  at  once  Hailed  and  interpreted  by 
many  as  measuring  the  degree  of  immunity  possessed 
by  the  patient.  The  same  error  was  made  when 

1  Bolduan,  Long  Island  Med.  Journal,  Vol.  i,  1907. 


IMMUNE  SERA 


some  time  later  the  bacteriolytic  substances  were 
discovered.  In  both  cases  it  was  soon  found  that 
these  were  but  accompaniments  of  greater  or  less 
significance  to  the  complex  phenomenon  of  immun- 
ity. When  we  consider  how  manifold  are  the  defen- 
sive agencies  which  the  animal  organism  possesses, 
and  how  very  complex  they  become  the  more  they 
are  studied,  we  shall  not  marvel  at  the  absence  of 
parallelism  between  the  clinical  course  of  the  disease 
and  the  opsonic  index.  There  is  little  doubt  that 
the  opsonic  indices  do  measure  a  certain  fraction  or 
phase  of  the  immunity  reaction;  we  do  not  believe 
that  they  replace  clinical  observations  in  measuring 
the  effect  of  immunizing  injections. 


VII.  SNAKE  VENOMS  AND  THEIR  ANTISERA 

Despite  the  fact  that  venomous  serpents  have 
excited  the  fear  and  interest  of  mankind  for  centuries 
it  is  only  very  recently  that  we  have  come  to  know 
anything  definite  about  their  poisons.  This  is 
perhaps  in  part  due  to  the  fact  that  Europe  possesses 
but  few  poisonous  snakes,  and  so  offered  little 
material  for  study.  Some  idea  of  the  importance 
of  the  subject  for  certain  countries,  however,  can  be 
seen  when  it  is  stated  that  in  India  more  than 
20,000  persons  annually  die  from  the  bite  of  the 
hooded  cobra.  It  was  quite  natural,  therefore,  that 
one  of  the  earliest  modern  researches  into  the 
nature  of  snake  venom,  that  of  Calmette,1  should 
have  come  from  that  country.  This  author  also 
found  that  he  could  produce  an  antitoxic  serum  by 
injecting  animals  with  the  snake  venom. 

The  Venoms.  —  Our  present  knowledge  of  snake 
venoms  and  their  antisera  is  due  largely  to  the 
researches  of  Flexner  and  Noguchi 2  and  of  Kyes 
and  Sachs.3  The  venoms  of  different  snakes  vary 

1  Calmette,   Annal.    Inst.    Pasteur,   Vol.    vi,    1892;   Comptes 
rend.  Soc.  Biol.,   1894. 

2  Flexner  and  Noguchi,  Journal  Exp.  Medicine,  1902,  et  seq. 
s  Kyes  and  Sachs.  See  in  Collected  Studies  on   Immunity, 

Ehrlich-Bolduan,  New  York,  1910. 


138  IMMUNE  SERA 

a  great  deal  in  their  toxic  properties,  and  this  is 
due  to  their  relative  contents  of  different  consti- 
tuents, as  follows:' — haemagglutinins,  haemolysin, 
hsemorrhagin,  and  neurotoxin.  The  first  two  act 
exclusively  on  the  blood  cells,  the  haemorrhagin  on 
the  endothelium  of  the  blood  vessels,  and  the 
neurotoxin  on  the  cells  of  the  central  nervous 
system.  The  last  named  causes  death  by  paralysis 
of  the  cardiac  and  respiratory  centers.  The  ven- 
oms of  the  cobra,  water-moccasin,  daboia  and 
some  poisonous  sea  snakes  are  essentially  neuro- 
toxic,  although  they  have  strong  dissolving  powers 
for  the  erythrocytes  of  some  animals.  In  study- 
ing the  haemolytic  powers  of  the  venoms  of  cobra, 
copperhead,  and  rattlesnake,  Flexner  and  Noguchi 
found  cobra  venom  to  be  the  most  haemolytic  and 
that  of  rattlesnake  the  least.  They  attribute  the 
toxicity  of  rattlesnake  poison  chiefly  to  the  action 
of  haemorrhagin.  The  venoms  of  the  water  mocca- 
sin and  the  copperhead  also  contain  haemorrhagin. 

Unlike  the  bacterial  toxins  the  action  of  the  snake 
venoms  is  preceded  by  no  appreciable  incubation 
period.  In  addition  to  this  the  poisons  are  very 
rapidly  absorbed.  Thus  Calmette  found  that  a  rat 
inoculated  into  the  tip  of  the  tail  could  not  be  saved 
by  amputating  the  tail  orie  minute  later.  Such 
animals  died  within  about  five  minutes  of  the  time 
required  for  control  animals. 

The  haemolysin  and  neurotoxin  and  perhaps  also 


SNAKE   VENOMS  AND   THEIR  ANTISERA      139 

the  other  cytotoxic  substances  of  venom  consist  of 
amboceptors  which  find  a  complement  in  the  body 
of  the  poisoned  animal.  Not  only  does  ordinary 
serum-complement  serve  for  activation,  but,  accord- 
ing to  Noguchi,1  the  fatty  acids  contained  in  the  red 
blood  cells  also  act  as  complement.  Lecithin  is 
also  able  to  reactivate  the  haemolysins  of  cobra 
venom,  forming,  according  to  Kyes,  a  "cobra- 
lecithid."  Recent  experiments  by  Manwaring,2 
however,  show  that  the  product  obtained  by  Kyes 
was  really  a  venom-free  lecithin  derivative  and  not 
a  "lecithid." 

Antivenins.  —  Calmette  was  the  first  to  produce 
an  antiserum  against  snake  venom,  utilizing  for 
this  purpose  rabbits.  He  began  with  injections  of 
Jv  of  a  fatal  dose,  and  injected  gradually  increasing 
doses  until  at  the  end  of  four  or  five  weeks  the 
animals  tolerated  double  a  fatal  dose.  By  con- 
tinuing the  treatment  he  finally  got  the  animals 
to  stand  80  fatal  doses  (40  mg.)  without  any 
reaction  whatever.  Five  drops  of  the  serum  of  such 
an  animal  neutralized  i  mg.  cobra  poison.  It 
has  been  found  that  anticobra  serum  protects 
against  the  neurotoxic "  components  of  other  snake 
venoms,  furthermore  against  scorpion  poison  and 
the  poison  of  eel  blood.  The  serum  also  contains 

1  Noguchi,  Journ.   Exper.  Medicine,  Vol.  ix,  1907. 

2  Manwaring,  Johns  Hopkins   Hospital    Bulletin,   September, 
1910, 


140  IMMUNE  SERA 

an  antihaemolysin,  but  no  antibody  against  haemor- 
rhagin  (of  the  rattlesnake).  It  is  therefore  without 
effect  on  rattlesnake  venom.  Antivenin  for  the 
latter  may  be  prepared  by  immunizing  goats  with 
corresponding  venoms  which  have  been  attenuated 
by  weak  acids.  Such  a  serum,  of  course,  possesses 
no  antineurotoxin  and  is  therefore  useless  against 
cobra  and  viper  venoms. 


VIII.     ANAPHYLAXIS 

Historical. — In  1898  Richet  and  Hericourt  showed 
that  when  dogs  were  injected  with  eel  serum  they 
not  only  failed  to  develop  an  immunity  against  this 
poison,  but  actually  became  more  susceptible. 
Subsequently  they  made  similar  observations  with 
a  toxin,  mytilo-congestin,  isolated  from  mussels. 
Richet  applied  the  term  "  anaphylaxis  "  to  this 
phenomenon  to  distinguish  it  from  immunization 
or  prophylaxis.  Arthus,  in  1903,  reported  that 
similar  effects  could  be  obtained  with  substances 
ordinarily  not  poisonous.  Thus  he  found  that  if 
rabbits  were  injected  with  horse  serum  they  were 
rendered  very  susceptible  to  a  second  injection 
made  after  an  interval  of  six  to  eight  days.  The 
second  injection  produced  severe  symptoms,  and 
sometimes  led  to  death  in  these  animals.  Little 
or  no  attention  was  paid  to  these  observations.  Fol- 
lowing a  statement  made  to  him  by  Theobald  Smith 
in  1904,  Ehrlich  caused  his  pupil,  Otto,  to  study 
why  guinea  pigs  which  had  been  injected  with 
toxin-antitoxin  mixtures  in  the  course  of  standard- 
ization of  diphtheria  antitoxin,  should  so  often  be 
killed  by  a  subsequent  injection  of  horse  serum. 

Independently  of  this  the  subject  was  being  investi- 

141 


1 42  IMMUNE   SERA 

gated  by  Rosenau  and  Anderson  in  the  Hygienic 
Laboratory.  Almost  simultaneously  with  the  ap- 
pearance of  these  studies  came  a  comprehensive 
monograph  on  the  serum  rashes  by  v.  Pirquet  and 
Schick,  and  this  fitted  in  so  well  with  the  labora- 
tory studies  of  Otto  and  of  Rosenau  and  Anderson 
that  a  great  deal  of  interest  was  aroused  in  this 
subject. 

The  Phenomenon. — As  a  result  of  all  the  work 
that  has  been  done  we  now  know  that  when  an 
animal  is  injected  with  an  alien  proteid,  there 
develops,  after  a  time,  a  specific  hypersusceptibility 
for  this  proteid.  After  a  definite  interval  if  the 
animal  is  given  a  second  injection  of  the  same 
proteid,  violent  symptoms  appear,  often  leading 
to  the  death  of  the  animal.  The  reaction  is  specific, 
so  that  animals  sensitized,  for  example,  to  horse 
serum,  manifest  little  of  no  hypersusceptibility 
to  other  sera.  It  is  possible,  however,  to  sensitize 
an  animal  to  several  proteids  simultaneously.  The 
sensitizing  dose  may  be  very  small — -even  as  little 
as  one  millionth  cubic  centimeter  of  horse  serum 
has  sufficed  to  render  guinea  pigs  sensitive.  A 
varying  length  of  time  must  elapse  after  the  sen- 
sitizing injection  before  the  animal  becomes  fully 
sensitized.  In  guinea  pigs  injected  with  small  doses 
of  horse  serum,  from  twelve  to  fourteen  days 
suffices.  With  larger  doses,  however,  the  time 
required  is  much  longer,  and  may  extend  over 


ANAPH  YLAXIS  143 

weeks  or  even  months.  The  hypersusceptibility 
is  transmitted  from  mother  to  offspring,  and  may 
also  be  passively  transferred  to  other  animals  by 
transferring  some  of  the  serum  of  the  sensitized 
animal  to  normal  animals.  Animals  recovering 
from  the  symptoms  induced  by  the  second  injection 
are  thereafter  no  longer  hypersensitive  to  the  pro- 
teid  employed,  but  are  immune.  This  immunity 
is  spoken  of  as  "  antianaphylaxis."  This  condi- 
tion can  also  be  brought  about  artifically  by  inject- 
ing the  animals  after  they  have  received  their 
sensitizing  injection  and  just  before  the  end  of 
the  anaphylactic  incubation  time,  with  compara- 
tively large  quantities  of  the  same  proteid.  Rosenau 
and  Anderson  have  shown  that  animals  may  be 
sensitized  by  feeding  them  with  the  proteid. 
Whether  this  has  any  practical  application  to  the 
clinical  use  of  specific  immune  sera  derived  from 
horses  in  persons  habitually  eating  horse  flesh  is  not 
known. 

Serum  Rashes. — Turning  our  attention  for  a 
moment  to  the  serum  rashes,  we  find  that  in  1874 
Dallera  reported  that  urticarial  eruptions  might 
follow  the  transfusion  of  blood.  Neudorfer  as  well 
as  Landois  also  refer  to  this  complication.  In  the 
year  1894  the  use  of  diphtheria  antitoxin  introduced 
the  widespread  practice  of  injecting  human  beings 
with  horse  serum.  In  the  same  year  several  cases 
were  reported  in  which  these  injections  were  fol- 


I44  IMMUNE  SERA 

lowed  by  various  skin  manifestations,  mostly  of 
an  urticarial  character.  Following  these  came 
a  great  mass  of  evidence  which  made  it  clear  that 
following  the  injection  of  antidiphtheric  serum 
these  sequelae  were  usually  comparatively  harmless. 
Heubner  in  1894  and  von  Bokay  somewhat  later 
expressed  the  opinion  that  these  manifestations 
were  due  to  other  properties  than  the  antitoxin 
in  the  serum,  and  this  has  proved  to  be  the  case. 
Johannessen  produced  the  same  effects  by  injecting 
normal  horse  serum.  It  has  been  shown  that  the 
skin  eruptions  and  other  symptoms  follow  in  direct 
proportion  to  the  amount  of  serum  injected,  a  fact 
which  has  led  to  the  concentration  of  the  sera  by 
getting  rid  of  the  non-antitoxic  proteid  constituents. 
In  their  exhaustive  study,  already  mentioned, 
v.  Pirquet  and  Schick  described  the  various  clinical 
manifestations  following  the  injection  of  horse  serum 
into  man,  and  gave  the  name  "  serum  disease  "  to 
the  symptom  complex.  The  principal  symptoms 
of  this  disease  are  a  period  of  incubation  varying 
in  length  from  eight  to  thirteen  days,  fever,  skin 
eruptions,  swelling  of  the  lymph  glands,  leucopenia 
joint  symptoms,  oedema,  and  albuminuria. 

Theories  of  Anaphylaxis. — It  was  difficult  tc 
account  for  the  long  period  of  incubation  in  the  pro- 
duction of  these  serum  rashes.  With  poisons 
capable  of  self -multiplication  (bacteria,  etc.),  this 
period  was  usually  referred  to  the  time  necessary 


ANAPHYLAXIS 

for  them  to  accumulate  in  sufficient  number 
and  virulence  to  produce  symptoms.  But  serum 
is  not  a  poison  capable  of  multiplication. 
Pfeiffer's  work  on  the  endo toxins  led  to  the  view 
that  the  antibodies  played  an  important  part  in 
bringing  on  the  symptoms  by  setting  free  the  endo- 
toxins.  The  results  of  these  observations  are  very 
closely  related  to  von  Pirquet  and  Schick's  explan- 
ation of  the  production  of  serum  disease.  The 
endotoxin  theory,  in  the  sense  of  bacteriolysis, 
naturally  cannot  be  applied  to  albuminous  sub- 
stances in  solution.  We  can  only  accept  it  in  the 
sense  that  by  means  of  the  reaction  between  the 
antibodies  and  the  antigen  the  poisonous  substance 
is  formed.  The  period  of  incubation,  both  in  serum 
rashes  and  in  bacterial  infections,  is  thus  readily 
understood,  for  it  is  at  once  apparent  that  the 
formation  of  antibodies  requires  time.  The 
general  idea  underlying  von  Pirquet  and  Schick's 
theory  of  serum  disease  is  that  the  injection  of  the 
horse  serum  into  man  causes  the  development  of 
specific  reaction  products  which  are  able  to  act  upon 
the  antigens  introduced.  These  antibodies  encoun- 
ter the  antigens,  i.e.,  some  of  the  serum  still  present 
in  the  body,  and  so  give  rise  to  a  poisonous  sub- 
stance. This  accounts  also  for  the  cases  of  "  imme- 
diate reaction"  described  by  von  Pirquet  and  Schick 
in  which  the  second  injection  of  a  serum  produces 
an  attack  of  serum  disease  without  any  period  of 


146  IMMUNE  SERA 

incubation.  Here  the  second  injection  comes  at  a 
time  when  the  accumulation  of  antibodies  is  at  its 
height.  It  has  been  claimed  that  this  explains  the 
cases  of  sudden  death  in  humans  following  injec- 
tions of  serum,  but  investigation  shows  that  most 
of  these  deaths  occurred  after  but  a  single  injection 
of  serum.  Moreover  in  most  of  them  such  conditions 
as  status  lymphaticus  sufficed  to  explain  the  fatal 
ending. 

This  theory  has  found  some  experimental  con- 
firmation from  the  work  of  Vaughan  and  Wheeler, 
who  have  been  able  to  prepare  a  number  of  split 
products  from  the  proteid  molecule,  some  of  which 
in  animals  give-  rise  to  a  symptom  complex  not 
unlike  that  of  typical  anaphylaxis. 

Gay  and  Southard  hold  a  somewhat  different 
view.  According  to  them  the  "  horse  serum  con- 
tains a  substance,  anaphylactin,  which  is  not  neu- 
tralized, and  is  eliminated  from  the  body  with  great 
slowness.  When  a  normal  guinea  pig  is  injected 
with  a  small  amount  of  horse  serum,  the  greater 
part  of  its  elements  are  rapidly  eliminated;  the 
anaphylactin,  however,  remains  and  acts  as  a 
constant  irritant  to  the  body  cells,  so  that  their 
avidity  for  the  other  assimilable  elements  of  horse 
serum  which  have  accompanied  the  anaphylactin 
becomes  enormously  increased.  At  the  end  of  two 
weeks  of  constant  stimulation  on  the  part  of  the 
anaphylactin,  and  of  increasing  avidity  on  the  part 


ANAPHYLAXIS 

of  the  somatic  cells,  a  condition  has  arrived  when 
the  cells,  if  suddenly  presented  with  a  large 
amount  of  horse  serum,  are  overwhelmed  in  the 
exercise  of  their  assimilating  functions,  and  func- 
tional equilibrium  is  so  disturbed  that  local  or 
general  death  may  follow."  According  to  this  view 
the  intoxication  caused  by  the  second  injection 
depends  upon  constituents  of  the  serum  eliminable 
by  the  animal  body. 

Allergy. — It  is  apparent  that  what  has  been  said 
concerning  the  production  of  anaphylaxis  in  re- 
sponse to  serum  injections  will  apply  also  to  bac- 
terial infections,  for  in  these  the  body  is  injected, 
as  it  were,  with  bacterial  proteids.  The  phenomena 
of  anaphylaxis  are  therefore  of  general  application  in 
immunity.  This  is  well  expressed  by  von  Pirquet,1 
who  calls  attention  to  the  fact  that  the  main  differ- 
ence between  a  normal  and  an  immune  individual 
is  one  relating  to  the  alteration  in  the  latter's  re- 
activity. He  speaks  of  this  alteration  as  "allergy" : 
from  ergeia,  reactivity,  and  allos,  altered,  meaning 
thereby  a  changed  reactivity  as  a  clinical  conception 
unprejudiced  by  bacteriological,  pathological  or 
biological  findings.  This  alteration  may  relate  to 
the  quality  and  quantity  of  the  symptoms  and  to 
their  rate  of  development.  Allergy  seems  to  be 
associated  more  with  some  infections  than  with 
others.  Experimentally  it  can  best  be  studied  by 

1  C.  E.  von  Pirquet,  Archives  of  Internal  Medicine,  Feb.,  191  r. 


148  IMMUNE  SERA 

observing  the  effect  of  cow-pox  inoculation  in 
primary  and  subsequent  vaccinations.  The  re- 
vaccinated  overcomes  the  whole  process  with  a  very 
slight  local  reaction  a  few  millimeters  in  size,  while 
the  person  vaccinated  the  first  time  shows  extensive 
local  inflammation,  fever,  and  other  general  symp- 
toms. If  the  reaction  is  studied  on  the  day  follow- 
ing the  vaccination,  we  shall  find  that  the  re- 
vaccinated  is  really  hypersensitive,  because  at  this 
time  the  first  vaccinated  does  not  show  any  reaction, 
while  the  revaccinated  responds  with  a  local  inflam- 
matory process.  In  tuberculosis,  glanders,  and 
other  infections  the  injection  of  extracts  of  the 
infecting  bacterium  (tuberculin,  mallein,  etc.)  pro- 
duces characteristic  local  and  general  symptoms, 
because  of  the  specific  hypersensitive  condition 
present  in  such  infections.  These  reactions  can 
therefore  be  employed  in  the  diagnosis  of  such  in- 
fections. The  symptoms  of  hay  fever,  and  of  urti- 
caria appear  to  be  merely  examples  of  proteid 
hypersensi  ti  ven  ess . 

Supposed  Relation  to  Precipitin  Action.  — 
Attempts  have  also  been  made  to  associate  the 
phenomena  of  anaphylaxis  with  the  action  of  pre- 
cipitins.  Hamburger  and  Moro  were  the  first  (1903) 
who  found  that  man  forms  precipitins  after  the 
injection  of  horse  serum.  Precipitin  was  present 
after  the  appearance  of  serum  rashes ;  therefore  they 
suggested  a  connection  between  serum  exanthem 


ANAPHYLAXIS 


149 


and  precipitin  formation,  without  looking  on  the 
precipitation  itself  as  the  cause  of  the  rash.  More 
recently  Doerr  anci  Russ,  as  the  result  of  experi- 
ments, hold  that  the  phenomena  of  anaphylaxis  are 
due  to  a  reaction  between  precipitins  attached  to 
the  tissue  cells,  and  the  precipitable  antigen.  The 
anaphylactic  shock  is  looked  upon  as  an  intracellu- 
lar  precipitin  reaction.  In  quantitative  investiga- 
tions these  authors  showed  that  the  amount  of 
anaphylactic  antibody  in  the  serum  of  rabbits 
was  always  parallel  to  its  precipitin  content.  It 
has  also  been  found  that  animals  which  do  not  form 
precipitins,  like  white  mice,  are  incapable  also  of 
forming  the  anaphylactic  antibody.  Against  the 
view  that  precipitins  have  anything  to  do  with 
anaphylaxis  in  man  is  the  fact  that  the  symptoms 
of  serum  disease  appear  within  eight  to  thirteen 
days  following  the  first  injection  of  horse  serum, 
whereas  it  requires  about  three  weeks  for  precipi- 
tins to  appear  in  the  blood  in  children  after  the 
injection  of  horse  serum.  Furthermore,  the  forma- 
tion of  precipitins  does  not  take  place  as  readily 
in  man  following  the  injection  of  horse  serum  as 
it  does  in  rabbits.  In  fact  von  Pirquet  found  that 
sometimes  even  after  the  injection  of  200  cc.  there 
was  no  production  of  precipitins.  Finally  it  may 
be  remembered  that  there  is  no  evidence  that  the 
precipitin  action  is  other  than  a  test-tube  phenom- 
enon, or  that  it  ever  occurs  in  vivo.  Friedemann 


1 50  IMMUNE  SERA 

has  shown  that  the  precipitates  produced  in  vitro 
will,  when  injected  intravenously  into  animals,  pass 
through  the  capillaries  without  harmful  effects. 

Pathology  of  Anaphylactic  Shock.  —  Acute  ana- 
phylactic  death  in  guinea-pigs  was  originally  attrib- 
uted to  asphyxia  of  central  origin.  Auer  and 
Lewis,1  however,  showed  that  the  asphyxia  is  due 
to  a  tetanic  contraction  of  the  bronchial  muscle, 
the  contraction  being  so  pronounced  that  the  lumina 
of  the  smaller  bronchi  are  occluded,  thus  preventing 
both  the  entrance  and  the  escape  of  air.  In  a 
recent  study  of  the  subject,  Schultz  and  Jordan2 
show  that  in  guinea-pigs  the  point  of  occlusion  is 
usually  just  beyond  the  place  where  the  secondary 
bronchi  leave  the  primary,  and  in  all  cases  at  points 
commanding  large  areas  of  lung  tissue.  At  this 
point  there  is  the  greatest  relative  (to  diameter  of 
lumen)  amount  of  smooth  muscle,  and  there  is  also 
normally  a  thicker  mucosa  and  greater  degree  of 
folding  of  the  same  relative  to  the  lumen.  The 
fatal  asphyxia  observed  in  guinea-pigs  is  therefore 
due  to  the  peculiar  anatomical  condition  of  the 
bronchial  tree  in  these  animals.  In  white  mice 
the  anaphylactic  reaction  shows  itself  by  increased 
peristalsis,  contractions  of  the  bladder,  increased 
irritability  of  the  skin,  etc.  The  respiratory  symp- 

1  Auer  and  Lewis,  Journal  Exp.  Medicine,  Vol.  xli,  1910. 

2  Schultz  and  Jordan,  Journal  Pharmacol.  and  Exp.   Thera- 
peutics, Vol.  ii,  March,  1911. 


ANAPHYLAXIS 

toms  are  absent.  This  is  clearly  because  the  mucosa 
of  the  bronchial  tree  is  nowhere  sufficiently  thick 
or  folded,  relative  to  the  amount  of  muscle  and  to 
the  diameter  of  lumen,  to  produce  occlusion  under  the 
amount  of  constriction  produced  by  the  contracting 
musculature.  The  recent  work  of  Schultz  shows 
that  serum  anaphylaxis  is  essentially  a  hyper- 
sensitization  of  smooth  muscle  generally. 

It  is  possible  that  the  occasional  occurrence  of 
severe  symptoms  and  even  of  death  in  man  follow- 
ing the  injection  of  serum  is  sometimes  due  to  an 
abnormal  development  or  condition  of  the  mucous 
membrane  and  smooth  muscle  of  the  bronchi. 
Some  support  is  given  to  this  view  by  the  more 
frequent  occurrence  of  these  disturbances  in  asth- 
matic individuals. 

Relation  of  Anaphylaxis  to  Serum  Therapy.— 
Returning  now  to  the  relation  of  the  experimental 
work  in  anaphylaxis  to  serum  therapy,  attention 
should  be  called  to  the  work  of  Steinhardt  and  Banz- 
haf,  who  show  that  the  anaphylactic  reaction  in 
rabbits  differs  considerably  in  character  from  that 
observed  in  guinea-pigs.  These  authors,  therefore, 
warn  against  utilizing  the  results  of  experiments  on 
guinea-pigs  without  reservation  for  the  interpreta- 
tion of  phenomena  observed  in  human  beings.  It 
is  probable  that  man  cannot  be  sensitized  in  the 
same  way  as  guinea  pigs,  the  most  susceptible  of 
the  laboratory  animals.  Children  have  in  numerous 


152  IMMUNE  SERA 

instances  been  injected  with  antidiphtheric  horse 
serum  at  short  and  long  intervals,  without,  so  far 
as  we  are  aware,  causing  death.  Certain  serums, 
for  example,  the  anti tubercle  serum  of  Maragliano 
and  the  antirheumatic  serum  of  Menzer,  are  habit- 
ually used  by  giving  injections  at  intervals  of  days 
or  weeks.  It  may,  of  course,  be  pbjected  that 
possibly  these  injections  are  so  spaced  as  to  produce 
antianaphylaxis.  If  a  person  had  once  before  had 
an  injection  of  horse  serum,  would  it  be  safe,  say 
some  months,  or  a  year,  or  several  years  later,  to 
give  him  another  injection  of  horse  serum?  Or  if 
a  child  had  been  immunized  against  diphtheria 
would  it  be  safe  to  repeat  the  injection  a  year 
later  if  the  child  were  again  exposed?  The  exper- 
ience of  clinicians  is  practically  unanimous  in  show- 
ing that  such  second  injections  need  not  be  feared. 
Even  if  the  results  obtained  in  guinea  pigs  were 
applicable  to  man,  a  subcutaneous  injection  in  man 
comparable  to  the  amount  required  to  produce 
sickness  in  a  guinea  pig  would  be  over  200  cc. 
To  date  about  twenty  cases  of  sudden  death  follow- 
ing the  injection  of  horse  serum  have  been  recorded 
in  the  literature,  and  while  this  undoubtedly  does 
not  represent  all  the  cases  that  have  occurred,  the 
total  number  is  insignificant  when  compared  to  the 
enormous  number  of  such  injections  already  made. 
In  New  York  City,  in  over  50,000  persons  injected, 
but  two  deaths  attributed  to  the  serum  injection 


ANAPHYLAXIS  !53 

have  occurred.  A  number  of  fatal  cases  have  been 
reported  in  asthmatic  individuals,  and  this  may  be 
borne  in  mind  when  about  to  make  serum  injec- 
tions. It  is  also  of  interest  to  know  that  Banzhaf 
and  Famulener  have  shown  that  chloral  in  large 
doses  will  prevent  the  anaphylactic  reaction  in 
sensitized  guinea  pigs.  Such  animals  after  the 
second  injection  are  immune  to  further  injections. 


IX.     INFECTION  AND  IMMUNITY 

Infection.  In  the  preceding  chapters  we  have 
studied  the  formation  and  mode  of  action  of  the 
various  antibodies.  Let  us  now  summarize  briefly 
our  knowledge  concerning  the  factors  involved 
in  infection  and  immunity.  An  infectious  disease 
is  one  caused  by  a  living  organism  wiiich  has  gained 
access  to  the  tissues  of  the  body.  A  study  of 
infection  and  immunity,  therefore,  embraces  a 
study  of  the  pathogenesis  of  these  organisms  on  the 
one  hand  and  of  the  defensive  agencies  of  the  body 
on  the  other.  So  far  as  the  invading  organism 
is  concerned,  we  know  that  this  may  remain  localized 
or  be  widespread  through  the  body.  The  absorption 
of  chemical  products  from  a  local  infection  may 
produce  general  symptoms.  This  is  known  as  an 
intoxication,  and  is  observed  in  cholera,  diphtheria, 
tetanus,  local  abscess,  etc.  In  general  we  apply 
the  term  '  pathogenic '  to  organisms  capable  of 
producing  disease,  but  it  must  be  borne  in  mind 
that  this  is  a  relative  term,  for  an  organism  patho- 
genic for  one  species  of  animal  need  not  necessarily 
be  pathogenic  for  another  species. 

The   Infecting  Agent. — In   studying  the  patho- 
genicity   of   various   bacteria,    it   is   apparent   that 

154 


*     INFECTION  AND  IMMUNITY  155 

we  can  distinguish  several  classes  of  organisms. 
One  class  is  characterized  by  the  secretion  of 
highly  toxic  soluble  substances,  both  in  the  living 
body  and  in  the  culture  fluid.  The  type  of  this 
class  is  the  diphtheria  bacillus.  Another  class  pro- 
duces highly  toxic  substances,  which  instead  of 
being  given  off,  remain  within  the  body  of  the 
bacterium.  These  poisons  may  be  demonstrated 
in  old  cultures  in  which  a  certain  amount  of  dis- 
solution ("  autolysis  ")  has  taken  place,  or  they 
may  be  obtained  by  mechanically  breaking  up  the 
bacteria  by  pressure  and  grinding.  These  sub- 
stances are  spoken  of  as  endotoxins,  and  are  liber- 
ated in  the  body  when  the  bacteria  are  disintegrated 
by  the  bacteriolytic  agencies.  The  type  of  this 
class  is  the  spirillum  of  cholera,  an  organism  which 
produces  a  powerful  endotoxin  and  which  very 
readily  undergoes  bacteriolysis.  In  addition  to 
these  two  classes  we  know  of  a  large  number  of 
bacteria  which  neither  secrete  a  highly  toxic  soluble 
substance  as  do  diphtheria  bacilli  nor  disintegrate 
as  readily  as  the  cholera  spirilla,  and  which  never- 
theless are  extremely  pathogenic.  Hiss  has  sug- 
gested that  many  organisms,  if  not  all,  secrete  sub- 
stances which  are  not  soluble  in  their  condition 
at  secretion,  but  which  are  susceptible  to  digestion 
in  the  animal  body.  These  substances  thus  become 
soluble  and  assimilable,  and  when  toxic  act  harm- 
fully on  the  body  cells.  Under  ordinary  circum- 


156  IMMUNE  SERA 

stances  these  substances  are  broken  up  within  the 
leucocytes  and  the  poisons .  thus  set  free  at  once 
neutralized  by  neutralizing  bodies  present  within 
the  cells.  According  to  this,  conception  the  leu- 
cocytes exercise  a  double  function,  one  bactericidal 
and  bacteriolytic,  the  other  a  poison-neutralizing 
one.  The  bactericidal  and  bacteriolytic  bodies 
appear  to  escape  from  the  leucocytes  quite  readily, 
and  can  be  demonstrated  in  the  blood  plasma;  the 
neutralizing  bodies,  on  the  other  hand,  do  not  appear 
to  be  given  off  from  the  cell.  It  is  obvious,  there- 
fore, that  the  bacterial  substances  may  be  broken 
up  in  the  blood  plasma,  and  from  them  may  thus 
be  liberated  a  poisonous  body.  When  this  poison- 
ous body  is  assimilated  in  sufficient  quantity  by  the 
higher  cells  of  the  animal  organism,  death  ensues, 
and  ensues  the  more  quickly  the  more  rapid  the 
process  of  liberation. 

In  discussing  allergy  it  was  pointed  out  that  the 
phenomena  of  anaphylaxis  should  also  be  applied  to 
bacterial  infections,  because  in  these  the  body  was 
treated  with  small  doses  of  bacterial  proteid.  As  a 
result  of  his  studies,  Friedberger  concludes  that  it  is 
unnecessary  to  assume  the  existence  of  specific  endo- 
toxins  in  bacteria  to  account  for  the  various  symp- 
toms seen  in  bacterial  infections.  By  repeatedly 
injecting  sensitized  animals  with  minute  doses  of 
sheep  or  horse  serum,  he  found  it  possible  to  produce 
all  manner  of  fever  curves  at  will,  merely  by  varying 


INFECTION  AND   IMMUNITY  157 

the  size  of  the  dose  and  the  interval  between  injec- 
tions. From  this  he  concludes  that  the  diversity 
of  clinical  symptoms  of  various  infectious  diseases 
can  readily  be  explained  on  the  assumption  of  but 
a  single  poison.  He  speaks  of  it  as  anaphylatoxin, 
and  regards  it  as  a  cleavage  product  of  proteid  of 
whatever  origin  introduced  parenterally.  Just  as  in 
enteral  digestion  uniform  cleavage  products  are 
formed  from  most  diverse  proteids,  so  he  believes 
that  in  the  parenteral  proteid  decomposition  lead- 
ing to  the  formation  of  anaphylatoxin,  a  uniform 
poison  is  produced.  Whether  or  not  in  addition  to 
the  anaphylatoxin  there  are  other  specific  poisons  for 
the  various  infectious  diseases  is  entirely  immaterial ; 
their  existence  has  not  been  proved1  and  the  as- 
sumption of  their  existence  is  unnecessary.  In  con- 
sidering the  diversity  of  the  clinical  symptoms  of 
various  infectious  diseases,  it  must  be  remembered 
that  the  various  species  of  bacteria  differ  in  their 
virulence  and  in  their  rate  of  multiplication,  and 
the  invaded  organisms  also  differ  considerably  in 
their  antibody  production.  All  these  factors  serve 
to  modify  the  clinical  picture.  According  to  Fried- 
berger  the  assumption  of  a  common  "anaphyla- 
toxin" is  only  apparently  in  contradiction  to  the 
well-known  law  of  specificity  of  the  infectious  dis- 
eases. In  the  infectious  diseases  it  is  not  the  poison 

1  This  applies  only  to  the  infectious  bacteria,  not  to  those  pro- 
ducing extra-cellular  toxins. 


158  IMMUNE  SERA 

which  is  specific,  but  only  the  mode  of  its  produc- 
tion. The  production  of  anaphylatoxin  requires  the 
action  of  antibodies;  the  mere  solution  or  disinte- 
gration of  bacteria  by  other  means  does  not  suffice. 
In  other  words,  a  definite  cleavage  of  the  proteid 
molecule  is  necessary.  The  anaphylatoxin,  there- 
fore, is  not  identical  with  Pfeiffer's  "endotoxins," 
though  perhaps  the  latter  may  be  the  mother  sub- 
stance from  which  the  anaphylatoxin  is  derived. 

Another  important  factor  in  pathogenesis,  accord- 
ing to  Bail,  is  the  ability  of  many  bacteria  to  pro- 
duce certain  neutralizing  substances,  not  directly 
injurious,  but  able  to  inhibit  or  neutralize  the  anti- 
bacterial activities  of  the  body.  These  substances 
Bail  calls  aggressins.  There  is  still  some  doubt 
whether  they  are  a  distinct  class  of  bacterial 
products.  Wassermann  and  Citron,  Doerr,  and 
others  regard  them  as  consisting  of  dissolved 
bacterial  substances,  extracted  endotoxins  and 
toxins. 

Resistance  Against  Infection. — The  ability  of 
an  animal  to  resist  the  effects  of  a  pathogenic 
organism  is  spoken  of  as  immunity,  and  may  be 
either  natural  or  acquired.  For  example,  it  is  well 
known  that  the  lower  animals  are  immune  against 
syphilis  and  gonorrhoea,  that  dogs  and  goats  are 
rarely  affected  with  tuberculosis,  and  that  man  is 
naturally  immune  against  chicken  cholera  and 
rinderpest.  These  are  instances  of  natural  im- 


INFECTION  AND  IMMUNITY  159 

muiiity.  Furthermore,  it  is  well  established  that 
with  certain  diseases  one  attack  usually  protects 
the  individual  for  life.  This  is  well  seen  in 
small-pox,  scarlet  fever,  and  measles.  Inasmuch 
as  the  individual  was  previously  susceptible, 
this  form  of  immunity  is  spoken  of  as  acquired 
immunity. 

Natural  Immunity. — It  is  seldom  that  natural 
resistance  is  absolute.  Young  animals  are  often 
susceptible  to  an  infection  against  which  adults  are 
resistant.  Thus  young  pigeons  are  readily  infected 
with  anthrax  while  older  pigeons  are  usually 
refractory.  Moreover,  the  resistance  of  animals 
toward  infections  against  which  they  are  relatively 
immune  can  often  be  lowered  by  artificial  means. 
Frogs  can  be  infected  with  anthrax  if  they  are  kept 
in  water  at  a  temperature  of  35°  C.  Conversely, 
chickens,  which  also  are  relatively  immune  to 
anthrax,  can  be  infected  if  they  are  chilled.  White 
rats,  which  are  ordinarily  resistant  to  anthrax 
infection,  become  susceptible  after  fatigue  or  when 
fed  on  an  exclusively  vegetable  diet. 

Ehrlich  believes  that  natural  immunity  is  some- 
times due  to  the  absence,  in  the  body  of  the  invaded 
animal,  of  suitable  receptors  for  the  virus.  After 
what  has  been  said  in  connection  with  the  side 
chain  theory,  it  is  obvious  that  the  virus  cannot 
exert  its  pathogenic  action  if  there  are  no  receptors 
whereby  it  is  anchored  to  the  body  cells.  More- 


160  IMMUNE  SERA 

over,  as  Ehrlich  points  out,  even  if  such  receptors 
are  present,  it  is  possible  for  the  animal  to  be  immune 
provided  the  receptors  are  situated  only  in  indif- 
ferent, vitally  unimportant  tissues.  In  some  of 
the  lower  animals  there  is  reason  to  believe  that  the 
toxin  of  tetanus  does  combine  with  such  tissue 
(Metchnikoff). 

Acquired  Immunity, — This  may  be  either  active 
or  passive,  and  either  form  may  be  acquired  naturally 
or  artificially.  As  examples  of  naturally  acquired 
active  immunity  wre  may  mention  the  immunity 
developed  by  one  attack  of  small-pox,  scarlet 
fever,  etc.  The  immunity  against  small-pox  con- 
ferred by  vaccination  is  an  example  of  artificially 
acquired  active  immunity;  so  is  the  preventive 
inoculation  with  bacterial  vaccine  against  typhoid 
fever.  The  best  illustration  of  artificially  acquired 
passive  immunity  is  the  injection  of  diphtheria 
antitoxin  into  humans,  while  the  transmission  of 
antitoxic  immunity  from  mother  to  offspring  is 
an  example  of  naturally  acquired  passive  im- 
munity. 

So  far  as  maternal  transmission  of  immunity  is 
concerned,  a  number  of  writers,  among  whom  may 
be  mentioned  Ehrlich,1  Anderson,2  and  Theobald 

1  See  Morgenroth's  article  in  Kolle  and  Wasserman's  Hand- 
buch,  Vol.  iv,  p.  784. 

2  Anderson,   Bull.   Hyg.   Lab.   U.   S.    Pub.   Health  and  Mar. 
Hosp.  Serv.,  No.  30. 


INFECTION  AND  IMMUNITY  .16 1 

Smith,1  noted  that  an  actively  immunized  female 
parent  may  transmit  antibodies  to  the  immediate 
young,  who,  receiving  the  immunity  passively,  soon 
lose  it  again.  The  male  parent  is  unable  to  trans- 
mit any  immunity.  In  his  classical  studies  with 
ricin  and  abrin,  Ehrlich  showed  that  lactation 
played  an  important  part  in  the  transmission  of 
immunity  from  female  mice  to  their  immediate  off- 
spring. By  immunizing  a  nursing  mother  mouse 
(after  the  birth  of  the  litter)  he  was  able  to  demon- 
strate the  transmission  of  immunity  to  swine  plague 
to  the  nursing  young.  Smith,  on  the  other  hand, 
in  his  experiments  with  guinea  pigs,  immunized 
against  diphtheria  toxin,  found  that  lactation  played 
no  appreciable  part  in  the  passive  immunity  of  the 
young.  Salge  nursed  infants  with  the  milk  of 
goats  which  had  been  immunized  against  diphtheria 
and  against  typhoid,  and  was  unable  to'demonstrate 
the  passage  of  antibodies  to  the  infants. 

In  contrasting  active  with  passive  immunization 
we  may  say  that  the  former  is  usually  more  effect- 
ive, more  lasting,  and  productive  of  a  general 
immunity  and  not  merely  of  one  particular  kind. 
It  is,  however,  sometimes  difficult  to  carry  out, 
may  involve  some  risk  to  the  patient,  and  takes 
time.  Passive  immunization,  on  the  other  hand, 
is  usually  productive  of  only  a  limited  kind  of 
immunity,  i.e.,  antitoxic,  bactericidal,  opsonic,  etc., 

1  Smith,  Jour.  Exper.  Med.,  Vol.  xi,  1909. 


1 62  IMMUNE  SERA 

and  therefore  is  often  ineffective.  Consisting,  as 
it  usually  does,  in  the  injection  of  an  alien  serum, 
passive  immunization  produces  an  immunity  of  but 
short  duration,  the  body  apparently  getting  rid  of 
the  alien  proteid  as  rapidly  as  possible.  The  great 
advantage  of  this  form  of  immunization,  however, 
is  its  convenience,  freedom  from  risk  to  the  patient, 
and  above  all,  the  fact  that  the  immunity  is  pro- 
duced instantaneously. 

Mechanism  of  Immunity. — Infection,  whether 
natural  or  artificial,  is  usually  followed  by  a 
remarkable  series  of  alterations  in  the  tissues  of 
the  infected  host.  Representing,  as  it  does,  all  the 
tissues  of  the  body,  it  is  natural  that  these  changes 
are  most  strikingly  exhibited  in  the  blood.  The 
alterations  vary,  however,  both  with  the  kind 
of  bacterium,  and  with  the  animal  species  involved. 
Against  the  true  toxins,  including  probably  the 
leucocidins  and  hasmolysins,  the  body  produces  anti- 
toxins; against  the  bacterial  bodies  it  directs  the 
action  of  the  leucocytes  and  the  lytic  combinations 
formed  by  the  union  of  amboceptor  and  comple- 
ment; against  the  so-called  aggressins  it  directs 
the  opsonins  and  perhaps  also  the  bacteriolysins. 

Before  leaving  the  consideration  of  the  reaction 
of  the  body  to  infection,  attention  should  be  called 
to  the  comprehensive  investigations  of  Opie.  This 
observer  showed  that  the  cells  which  accumulate  in 
response  to  an  irritant  contain  enzymes,  the  enzyme 
of  the  polynuclear  leucocytes  resembling  trypsin 


INFECTION  AND  IMMUNITY  I62a 

and  the  enzyme  of  the  macrophages  resembling 
pepsin  in  its  action.  The  blood  serum,  on  the 
other  hand,  contains  an  antienzyme.  The  varying 
relation  existing  between  these  enzymes  and  the 
antienzymes  serves  to  explain  how  the  same  irritant 
in  the  same  quantity  may  cause  two  different  types 
of  inflammation.  This  is  well  illustrated  by  the 
following  experiment  made  by  Opie:1  If  a  small 
quantity  of  turpentine  is  injected  into  the  sub- 
cutaneous tissue  of  dog,  a  large  fluctuating  abscess 
filled  with  creamy  pus  is  formed  within  four  days; 
there  is  a  widespread  undermining  of  the  skin. 
The  same  quantity  of  turpentine  injected  into  the 
pleural  cavity  causes  a  serofibrinous  inflammation 
which  undergoes  resolution  so  that  the  pleural 
cavity  is  restored  to  its  normal  condition  after  about 
ten  days;  there  is  no  destruction  of  tissue  and  a 
scar  is  not  formed.  In  the  subcutaneous  tissue 
only  a  small  amount  of  oedematous  exudate  can 
accumulate;  the  undiluted  irritant  causes  active 
migration  of  leucocytes  so  that  the  antibody  of  the 
exuded  serum  is  soon  overbalanced  by  the  enzyme 
set  free  by  disintegrated  pus  cells.  In  the  pleural 
cavity,  on  the  contrary,  a  large  quantity  of  serum 
quickly  accumulates  and  the  exudate  is  sero- 
fibrinous instead  of  purulent;  the  antienzyme  it 
contains  is  capable  of  holding  in  check  the  enzyme 

1  E.  L.  Opie,  Lecture  before  the  Harvey  Society,  New  York, 
Feb.  1910.     The  Harvey  Lectures,  J.  B.  Lippincott  Co.     1910. 


l62b  IMMUNE  SERA 

of  the  accumulated  leucocytes.  If  a  bit  of  the 
fibrinous  exudate  is  suspended  in  the  exuded 
serum,  it  is  preserved  intact.  Nevertheless,  by 
repeated  injection  of  turpentine  at  short  intervals 
into  the  pleural  cavity,  accumulation  of  leucocytes 
can  be  prolonged  so  that  finally  a  condition  is 
produced  in  which  antienzyme  can  no  longer 
restrain  the  enzyme.  The  softened  fibrin  of  such 
an  exudate  quickly  disintegrates  in  the  serum  of 
the  exudate.  These  observations,  as  Opie  points 
out,  help  to  explain  how  the  typhoid  bacillus  pro- 
duces abscesses  in  certain  situations  such  as  the 
kidney  and  bone;  how  the  pneumococcus,  which 
rarely  causes  abscess  of  the  lung,  in  which  condi- 
tions are  somewhat  similar  to  those  within  the  pleural 
cavity,  may  cause  suppuration  in  other  localities, 
such  as  the  middle  ear,  or  in  the  subdural  space,  etc. 
In  addition  to  the  antibodies  already  mentioned, 
the  animal  body  produces  agglutinins  and  precip- 
itins  directed  against  the  invading  bacteria,  but 
the  relation  of  these  antibodies  to  immunity  is  not 
at  all  clear.  So  far  as  the  action  of  the  agglutinins 
is  concerned,  we  have  already  pointed  out  (on  page 
36)  that  this  appears  to  have  no  destructive  effect 
on  the  agglutinated  organisms.  Moreover,  while  ag- 
glutination is  often  observed  to  precede  lysis,  there  is 
no  reason  to  believe  it  a  necessary  factor  in  the  lytic 
process,  nor  even  an  aid  thereto.  Whether  this  list 
exhausts  the  number  of  serum  antibodies  is  doubtful. 


INFECTION  AND  IMMUNITY  163 

The  antibody  content  of  the  serum,  ^wever,  is 
not  always  the  same  as  that  of  the  blood  plasma. 
Thus  Gengou,  by  collecting  the  plasma  in  vaselined 
tubes,  found  it  often  to  be  almost  devoid  of  bac- 
tericidal power,  while  the  corresponding  serum  was 
capable  of  destroying  large  numbers  of  microor- 
ganisms. In  these  cases,  it  is  evident,  we  cannot 
regard  plasma  destruction  of  bacteria  as  the  impor- 
tant factor  in  immunity.  Moreover,  in  the  case  of 
bacteria  containing  considerable  quantities  of  endo- 
toxin,  it  is  conceivable  that  plasma  destruction  of 
bacteria  may  even  do  considerable  harm  by 
causing  an  enormous  liberation  of  endotoxin.  This 
point  is  perhaps  of  practical  importance  as  contra- 
indicating  the  use  of  bacteriolytic  sera  in  the 
curative  treatment  of  certain  infections. 

From  what  has  been  said  it  is  evident  that  the 
exact  mechanism  of  immunity,  at  least  so  far  as 
most  infections  are  concerned,  is  still  very  obscure. 
Like  most  biological  phenomena,  the  deeper  we 
analyze  the  problem,  the  more  complex  and  more 
marvelous  it  becomes.  Enough  has,  however,  been 
presented  to  show  some  of  the  difficulties  to  be 
overcome  and  the  method  of  attacking  the  sub- 
ject. 

Relation  of  Anaphylaxis  to  Immunity. — We  have 
already  discussed  the  relation  of  anaphylaxis  to 
infection  and  may  now  take  up  briefly  its  relation 
to  immunity.  We  know  that  the  subcutaneous, 


164  IMMUNE  SERA 

intraperitoneal,  or  intravenous  introduction  of  alien 
proteid  is  followed  by  the  formation  of  antibodies; 
at  the  same  time  it  can  readily  be  shown  that  no 
antibodies  develop  after  the  oral  introduction  of 
milk,  eggs,  or  even  of  raw  meat.  In  other  words, 
there  is  a  marked  contrast  in  the  behavior  of  the 
body  between  the  enteral  and  the  parenteral  intro- 
duction of  proteid.  In  the  former  the  proteid  is 
acted  on  by  the  gastric  and  intestinal  juices  (pepsin, 
trypsin,  and  enterokinase) .  These  so  break  down 
the  proteid  molecule  that  it  loses  its  species  identity. 
After  this,  absorption  takes  place,  and  with  it  there 
is  a  synthesis,  or  rearrangement,  of  the  molecule 
whereby  it  is  built  up  into  the  specific  proteid  of 
the  body.  Under  normal  conditions  it  is  im- 
possible to  produce  specific  antibodies  by  feeding 
alien  proteid.  Precipitins  have,  however,  been  pro- 
duced by  overfeeding  animals  with  large  quantities 
of  alien  blood.  When  proteid  is  introduced  paren- 
terally  it  gives  rise  to  the  formation  of  specific 
antibodies,  and  thus  to  the  state  of  anaphylaxis. 
The  term  anaphylaxis  is  unfortunate,  for  the  con- 
dition is  not  always  opposed  to  immunity.  Von 
Pirquet,  it  will  be  remembered,  called  attention  to 
the  altered  reactivity  during  the  anaphy lactic  state. 
We  must  not  lose  sight  of  the  fact  that  the  symp- 
toms of  anaphylaxis  are  brought  on  when  sensitized 
animals  are  subsequently  injected 'with  relatively 
large  quantities  of  the  same  proteid.  Following 


INFECTION  AND   IMMUNITY  165 

such  an  injection  there  is  a  sudden  liberation  of 
large  amounts  of  toxic  material.  The  parent eral 
introduction  of  large  quantities  of  alien  proteid 
must,  however,  be  very  exceptional  under  natural 
conditions.  The  number  of  bacteria  primarily 
involved  in  an  infection  certainly  represents  but  a 
very  small  amount  of  alien  proteid.  If  the  body  is 
in  the  condition  of  allergy  (anaphylaxis)  at  the 
time  of  infection  it  will  be  able  to  respond  more 
quickly  than  otherwise  and  perhaps  destroy  the 
invaders.  Under  these  circumstances  it  is  con- 
ceivable that  the  condition  is  really  an  immunity 
reaction.  Looking  at  the  entire  question  broadly 
we  may  regard  the  mechanism  which  lies  at  the 
bottom  of  the  phenomenon  of  anaphylaxis  as  a 
useful  contrivance  which  enables  the  organism  to 
rid  itself  of  alien  proteid,  both  organized  and  un- 
organized, which  has  been  introduced  parent erally. 
Immunity  Reaction  on  the  Part  of  Bacteria. — It 
may  be  well  at  this  point  to  call  attention  to  a  view 
advanced  by  Welch  some  years  ago.  According  to 
this  it  is  reasonable  to  suppose  that  just  as  the 
animal  body  produces  antibodies  against  an  invad- 
ing organism,  so  does  the  latter,  owing  to  the  action 
of  the  body  fluids,  produce  antibodies  directed 
against  the  tissues  of  the  invaded  body.  In  this 
way  the  infecting  organism  would  be  adapting  itself 
to  unfavorable  surroundings,  and  this  we  know  it 
often  does.  It  is  certain  that  the  animal  body  often 


1 66  IMMUNE  SERA 

successfully  overcomes  an  infectious  disease  without 
entirely  overcoming  the  infecting  bacteria.  This  is 
well  shown  by  what  we  call  chronic  germ  carriers. 
Deutsch  regards  the  increase  in  virulence  brought 
about  by  successive  passage  of  a  bacterium  through 
a  susceptible  animal  as  representing  an  immunity 
developed  by  the  bacterium  against  the  anti- 
bacterial agencies  of  the  body. 

Atrepsy. — Ehrlich  has  investigated  this  phe- 
nomenon in  the  case  of  trypanosomes.  He  found 
that  a  monkey  which  had  been  infected  with  a 
particular  strain  of  trypanosome  and  then  cured 
by  means  of  chemo- therapeutic  agents,  when 
tested  with  the  original  strain  was  not  immune, 
the  disease  reappearing  after  a  long  incubation. 
If  mice  were  inoculated  with  blood  from  the  diseased 
animal,  i.e.,  with  blood  containing  trypanosomes, 
they  became  infected  and  died.  Curiously,  how- 
ever, if  the  trypanosomes  were  first  removed  from 
this  monkey  blood,  it  was  found  that  the  serum 
was  able  to  kill  the  original  strain  of  trypanosomes. 
This  showed  that  the  trypanosomes  had  undergone 
some  change  in  the  body  of  the  monkey;  they 
differed  from  the  original  strain  in  their  behavior 
toward  the  serum;  they  had  become  "  serum-fast." 
Similar  observations  were  made  at  the  same  time 
by  Kleine,  and  recently  also  by  Mesnil. 

In  explanation  of  this  adaptation,  Ehrlich  sug- 
gests that  certain  particular  receptors  of  the  para- 


INFECTION  AND  IMMUNITY  i6f 

site  are  concerned  entirely  with  the  parasite's  nutri- 
tion. Owing  to  the  destruction  brought  about  by 
the  chemical  agent,  some  of  these  receptors  pass 
into  the  monkey's  body,  and,  acting  as  antigens, 
excite  the  production  of  antibodies  directed  against 
these  particular  receptors.  When  living  parasites 
are  brought  into  contact  with  this  antibody,  either 
in  vitro  or  in  vivo,  the  antibody  is  anchored  by 
the  parasites.  As  a  result  of  this  occupation  of 
its  receptors,  the  parasite  undergoes  a  biological 
alteration  which  consists  in  the  disappearance  of 
the  original  receptor  group  and  its  replacement 
by  a  new  group.  Ehrlich's  researches  lead  him  to 
believe  that  the  antibody  has  merely  an  anti- 
nutritive  action,  blocking  the  nutrireceptor  of  the 
parasite  and  so  bringing  about  starvation.  The 
parasite  thus  develops  immunity  by  getting  rid 
of  certain  of  its  nutrireceptors,  and  replacing  them 
with  different  ones.  This  form  of  immunity  Ehrlich 
speaks  of  as  "  atrepsy,"  while  the  antibodies  de- 
veloped against  the  nutrireceptors  he  terms  "  atrep- 
sins."  A  somewhat  different  example  of  atrepsy 
is  the  following:  Bird-pox,  virulent  for  both  fowl 
and  pigeon,  if  passed  through  the  pigeon  becomas 
completely  avirulent  for  the  fowl.  Ehrlich  believes 
that  the  parasite  in  passing  through  the  pigeon  has 
to  assimilate  substances  different  from  those  assim- 
ilated in  its  passage  through  the  fowl.  There- 
fore that  part  of  the  receptors  which  deals  with  the 


1 68  IMMUNE  SERA 

nutritive  substances  in  the  fowl's  organism  is  not 
in  use  during  the  passage  through  the  pigeon  and 
may  become  atrophied,  so  that  on  the  parasite 
being  transferred  back  to  the  fowl,  supposing  one 
of  the  specific  constituents  of  fowls  to  be  neces- 
sary for  its  proliferation,  it  would  no  more  be 
able  to  grow.  We  have,  therefore,  a  loss  of  cer- 
tain receptors  which  are  absolutely  necessary  for 
nutrition. 

Ehrlich  suggests  that  probably  the  majority  of 
so-called  non-pathogenic  micro-organisms,  if  intro- 
duced into  an  animal's  body,  perish  by  this  mechan- 
ism. It  is  not  necessary  to  assume  the  presence 
of  special  poisons  in  the  body,  it  suffices  to  suppose 
that  the  bacteria  in  question  do  not  find  the  needful 
means  of  existence  in  the  body  and  therefore  cannot 
multiply.  They  thus  fall  a  prey  to  the  phagocytes 
which  destroy  the  invaders  in  a  non-specific 
manner. 


X.     BACTERIAL  VACCINES 

Historical.— Early  in  the  eighteenth  century 
attention  was  called  to  the  fact  that  in  Oriental 
countries  individuals  were  immunized  against  small- 
pox by  inoculating  them  with  a  little  small-pox 
virus  under  the  skin.  In  1796  Jenner  showed  that 
similar  immunity  could  be  produced  by  inoculating 
the  virus  of  cow-pox,  and  this  procedure  was  free 
from  the  dangers  that  attended  small-pox  inocu- 
lations. Following  the  discovery  of  the  specific 
microbe  of  anthrax,  attention  was  directed  to  the 
problem  of  combating  this  disease.  Pasteur,  who 
had  been  greatly  impressed  with  Jenner's  work 
with  cow-pox,  felt  that  attempts  should  be  made 
to  produce  a  mild  attack  of  the  disease,  and  that 
this  would  then  protect  against  a  virulent  infection. 
After  considerable  experimental  labor  he  devised 
the  plan  of  inoculating  animals  with  cultures  of 
anthrax  which  had  been  attenuated  by  being  grown 
at  high  temperatures,  43°  C.  These  animals  had 
a  mild  attack  of  the  disease  from  which  they  soon 
recovered,  and  then  were  resistant  to  infection  with 

virulent  virus.     Soon  after  this,   inspired  by  Pas- 

169 


1 70  IMMUNE  SERA 

teur's    work,    successful    vaccines  *    were    prepared 
against  chicken  cholera  and  swine  plague. 

The  discovery  of  diphtheria  antitoxin  in  1893  by 
v.  Behring  marked  the  beginning  of  the  search  for 
specific  sera,  and  it  was  not  long  before  a  number 
of  such  were  produced  and  employed  clinically. 
The  use  of  sera  for  therapeutic  purposes  was  very 
attractive,  because  it  was  possible  to  have  some 
animal,  like  the  horse,  manufacture  the  antibodies, 
and  one  needed  then  merely  to  transfer  the  animal's 
immunity  to  the  patient  by  injecting  some  of  the 
animal's  serum.  Clinical  trials,  however,  soon 
showed  that  most  of  these  sera  had  little  thera- 
peutic value,  and  subsequently  laboratory  experi- 
ments disclosed  a  large  number  of  difficulties  in 
their  practical  application.  After  what  has  been 
said  under  haemolysins  and  bacteriolysins  it  will 
be  unnecessary  to  dwell  on  these  difficulties.  Among 
them  is  the  problem  of  providing  sufficient  com- 
plement, the  determination  of  the  optimum  dose 
so  as  to  avoid  the  parodoxical  results  known  as 
the  Neisser-Wechsberg  phenomenon,  the  ability 
of  producing  really  effective  antibodies,  and  finally 
the  question  whether  immunity  in  a  given  case  is 
really  directly  due  to  the  presence  of  these  anti- 
bodies in  the  serum. 

1  The  French  have  long  used  the  term  "vaccin"  to  denote 
any  virus  which  is  used  for  immunization,  and  that  is  the 
sense  in  which  the  term  is  used  here.  There  is,  of  course, 
nothing  of  the  cow,  vacca,  about  them. 


BACTERIAL  VACCINES  171 

In  the  past  few  years  it  has  become  more  and 
more  apparent  that  the  limitations  of  serum  therapy, 
at  least  in  the  great  majority  of  infectious  diseases, 
are  at  present  almost  insuperable.  Attention  was 
therefore  again  turned  to  treatment  by  active 
immunization.  It  was  perhaps  only  natural,  in 
view  of  his  discoveries  in  fermentation,  that  Pasteur 
should  have  believed  that  the  production  of  immu- 
nity required  the  action  of  the  living  virus.  He 
therefore  vigorously  combated  the  idea  that  im- 
munity could  be  brought  about  by  means  of  dead 
virus,  or  of  lifeless  products  of  growth  of  the  virus. 
Touissant,  as  far  back  as  1880,  had  held  out  for 
the  latter  possibility,  but  the  imperfections  of 
his  technique  were  such  that  his  views  were  not 
accepted.  To  Salmon  and  Smith  of  this  country 
belongs  the  honor  of  first  clearly  demonstrating 
the  possibility  of  immunization  writh  dead  cul- 
tures. 

Methods  of  Active  Immunization. — Active  im- 
munization can  be  carried  out  in  several  ways: 

(i)  By  means  of  living  cultures  of  the  virus. 
Usually  the  cultures  are  attenuated,  but  there  are 
some  exceptions. 

A  number  of  different  procedures  may  be  employed 
to  attenuate  the  virus.  Thus,  by  drying,  as  is  done 
with  rabies  virus  in  the  Pasteur  treatment;  or  by 
growing  the  virus  at  a  temperature  unsuited  for  the 
development  of  virulence,  as  is  done  in  the  case  of 


172  IMMUNE  SERA 

anthrax;  or  by  passing  the  virus  through  a  less  sus- 
ceptible animal,  as  is  done  in  vaccination  against  small- 
pox; or  by  means  of  chemicals  such  as  the  addition 
of  iodine  solution  to  diphtheria  toxin,  as  was  formerly 
done  by  Behring;  or  by  means  of  heat,  as  was  also 
formerly  done  with  diphtheria  toxin. 

(2)  By  means  of  dead  cultures  of  the  virus.     The 
cultures  can  be  killed  either  by  heat  or  by  the  use 
of  chemicals. 

(3)  By  the  so-called   "  combined  method,"  i.e., 
by  first  administering  a  dose  of  the  specific  immune 
serum  and  subsequently  the  virus.     This  method 
has  been  used  in  typhoid  fever,  cholera,  and  plague. 

(4)  By  means  of  the  products  of  autolysis  of  the 
cultures.     This  has  also  been  used  in  typhoid  fever, 
and  seems  to  possess  certain  advantages    over    the 
use  of  native  cultures. 

(5)  By  means    of    various    combinations   of    the 
preceding    methods. 

The  choice  of  these  various  methods  of  immuni- 
zation depends  on  the  nature  of  the  infecting  virus. 
With  some  infections  dead  cultures  apparently  are 
able  to  cause  the  production  of  full  protective  pow- 
ers, while  in  other  infections  the  body  seems  to 
require  a  greater  stimulus.  In  these,  the  use  of 
attenuated  living  cultures  may  bring  about  the 
desired  immunity.  Finally  there  are  infections 
in  which  nothing  short  of  fully  virulent  cultures 
seems  to  bring  about  the  development  of  sufficient 


BACTERIAL  VACCINES  173 

immunity.  In  these  cases  it  is  necessary  to  first 
prepare  the  way  by  the  use  of  dead  or  of  attenuated 
cultures. 

Treatment  with  Vaccines. — The  treatment  of 
infections  by  means  of  active  immunization  has 
been  greatly  stimulated  by  the  work  of  Wright, 
who  has  published  favorable  results  in  a  large 
number  of  infections.  Already  several  hundred 
thousand  persons  have  been  actively  immunized 
against  cholera,  and  large  bodies  of  troops  have 
been  immunized  against  typhoid  fever.  Until 
recently  the  method  found  application  particu- 
larly in  the  prophylactic  immunization  of  persons 
liable  to  be  exposed  to  infection.  At  the  present 
time,  however,  owing  largely  to  the  efforts  of 
Wright,  the  method  has  come  to  be  used  for  cura- 
tive purposes,  i.e.,  for  infections  already  in  progress. 
This  author  has  clearly  formulated  the  conditions 
in  which  he  thinks  this  form  of  treatment  is  indi- 
cated, and  he  has  also  devised  methods  for  the 
more  exact  determination  of  doses  than  were 
formerly  in  use. 

In  the  employment  of  bacterial  vaccines,  one 
must  constantly  keep  in  mind  the  nature  of  the 
bacterium  with  which  one  is  working,  and  the  kind 
of  immunity  one  wishes  to  bring  about.  Every- 
thing depends  on  the  way  in  which  the  vaccine  is 
prepared.  With  bacteria  making  considerable 
quantities  of  a  toxin,  it  will  be  necessary,  if  we 


174  IMMUNE  SERA 

wish  to  immunize  against  this  toxin,  to  grow  the 
culture  for  the  requisite  length  of  time  and  under 
the  proper  conditions  for  producing  the  toxin. 
In  the  case  of  bacteria  possessing  certain  endo- 
toxins,  it  may  be  necessary  to  let  the  cultures 
autolyze,  so  as  to  set  these  substances  free,  or  the 
bacteria  may  be  crushed  and  ground  for  the  same 
purpose.  On  the  other  hand,  we  may  wish  to  use 
these  bacteria  for  producing  a  specific  agglutinat- 
ing serum.  In  that  case  we  often  try  to  avoid 
injecting  these  toxic  substances.  Our  entire  pro- 
cedure might  then  have  to  be  quite  the  reverse  of 
what  has  just  been  indicated. 

The  Vaccines. — Wright's  method  of  preparing  a 
staphylococcus,  typhoid,  streptococcus,  or  gono- 
coccus  vaccine,  is  as  follows: 

Several  streak  slant  agar  cultures  are  planted  and 
incubated  for  twenty  to  twenty-four  hours.  The 
cultures  are  then  washed  off  with  normal  salt  solu- 
tion, using  from  one  to  several  cc.  for  each  culture. 
These  suspensions  are  next  heated  to  55°  C.  in 
order  to  kill  the  bacteria,  and  are  then  standard- 
ized. By  this  is  meant  determining  the  number  of 
organisms  per  cc.,  for  Wright  always  used  definite 
numbers  of  bacteria  in  his  inoculations.  This 
standardization  is  readily  accomplished  by  means 
of  the  method  devised  by  Wright,  which  is  as  fol- 
lows :  From  a  finger  prick  a  drop  of  blood  is  sucked 
up  in  a  capillary  tube  to  a  mark  made  at  any  con- 


BACTERIAL  VACCINES  175 

venient  point  with  a  wax  pencil.  Next  an  equal 
amount  of  the  bacterial  suspension  is  drawn  into 
the  tube,  allowing  a  tiny  air-bubble  to  intervene. 
The  two  fluids  are  then  expelled  on  a  glass  slide, 
and  thoroughly  mixed  by  sucking  back  and  forth 
a  number  of  times.  After  this  has  been  done  the 
mixture  is  spread  in  the  ordinary  way  of  making 
blood  smears.  If  these  blood  smears,  after  stain- 
ing, are  examined  with  a  microscope  having  a  ruled 
eye-piece,  it  is  a  simple  matter  to  determine  the 
ratio  of  bacteria  to  blood  cells.  Taking  the  red 
blood  cells  as  5,000  million  per  cc.,  one  calculates 
the  number  of  bacteria  per  cc.  In  practice  it  is 
advisable  to  so  dilute  the  bacterial  suspension  that 
the  dose  to  be  injected  is  contained  in  about  one 
cc.  of  fluid.  Finally  \  per  cent,  of  carbolic  acid 
is  added  as  a  preservative.  Such  a  suspension  is  a 
"  bacterial  vaccine."  It  goes  without  saying  that 
the  vaccines  should  be  tested  by  means  of  cultures 
to  insure  sterility,  and  that  contaminations  should 
be  excluded  by  means  of  microscopical  exami- 
nation. 

Doses. — So  far  as  doses  are  concerned,  these  vary 
with  different  bacteria,  and  also  according  to  the 
indications,  opsonic  or  clinical.  The  ordinary  dose 
for  the  staphylococcus  vaccine  is  from  200,000,000 
to  1,000,000,000  organisms;  for  the  streptococcus 
it  is  from  50  to  75  or  100,000,000,  and  for  typhoid 
from  750,000,000  to  1,000,000,000  bacteria.  All 


Ty6  IMMUNE  SERA 

the  injections  are  given  subcutaneously,  and  it  is 
well  to  repeat  the  injections  every  three  or  four 
days. 

Results. — The  clinical  results  obtained  by  means 
of  bacterial  vaccines  have  varied.  There  seems 
considerable  agreement  on  the  part  of  most  observers 
that  certain  localized  infections,  such  as  acne,  mul- 
tiple boils,  etc.,  usually  respond  remarkably  well 
with  this  method  of  treatment.  In  the  treatment  of 
bone  tuberculosis  the  results  are  not  so  harmonious, 
and  in  the  treatment  of  general  infections  many 
failures  have  been  reported.  There  is  no  doubt, 
however,  that  treatment  by  means  of  bacterial 
vaccines  is  a  valuable  addition  to  our  therapeutic 
armamentarium . 


XL     LEUCOCYTE  EXTRACTS  IN  THE  TREAT- 
MENT OF   INFECTIONS 

Theory. — Attention  has  already  been  called  to 
Hiss's  view  concerning  the  role  of  leucocytes  in 
combating  infections.  Believing  that  the  phagocytic 
power  of  leucocytes  of  persons  suffering  from  infec- 
tions to  be  less  than  that  of  leucocytes  of  the  normal 
individual,  Hiss  was  led  to  extract  these  cells  with 
a  view  to  utilizing  their  neutralizing  and  other  pro- 
tective substances  in  readily  diffusible  form.  By 
this  means  it  was  thought  possible  to  furnish  to  the 
infected  organism  such  assistance  as  would  enable 
its  phagocytic  cells  to  properly  protect  the  various 
tissues  from  poisons  elaborated  from  the  invading 
bacteria. 

Preparation  of  the  Extracts. — In  the  preparation  of 
the  extract,  double  pleural  inoculations  of  aleuronat  l 
suspensions  are  made  into  rabbits.  After  24  hours 
the  rabbits  are  killed  and  the  turbid  fluid  collected 
from  both  pleural  cavities.  The  quantity  obtained 
varies  from  about  30  to  60  cc.  The  fluid  is  quickly 
centrifuged  and  the  serum  decanted.  The  cells  are 

1  Aleuronat,  a  vegetable  product  similar  to  gluten,  is  pre- 
pared by  Hundhausen,  in  Hamm,  Westphalia,  Germany,  and 
is  supplied  in  packages  containing  100  grams.  The  suspensions 
are  prepared  with  thin  starch  paste  and  boiled. 

177 


1 78  IMMUNE  SERA 

then  thoroughly  emulsified  in  distilled  water,  using 
about  as  much  water  as  the  volume  of  serum  orig- 
inally poured  off,  and  the  mixtures  allowed  to 
stand  for  a  few  hours  at  37°  C.  This  more  or  less 
autolyzed  fluid  is  used  for  the  injections,  and  the 
dose  employed  varies  from  5  to  15  cc.,  repeated 
several  times. 

Application  and  Results. — In  their  work,  clinical 
and  experimental,  Hiss  and  Zinsser  1  thought  they 
saw  little  indication  of  immediate  bactericidal 
power  possessed  by  the  leucocyte  extract,  but  that 
the  results  pointed  rather  to  a  marked  power  on 
the  part  of  the  extract  to  reduce  the  purely  tox- 
semic  manifestations  in  infected  subjects.  Favorable 
clinical  results  have  been  reported  by  these  authors 
in  cerebrospinal  meningitis,  lobar  pneumonia,2  and 
other  infections,  and  while  the  data  are  still  too 
scanty  to  justify  definite  conclusions  as  to  the 
value  of  this  treatment,  enough  has  been  done  to 
warrant  further  careful  clinical  investigations  along 
this  line. 

1  Hiss  and  Zinsser,  Journal  Med.   Research,  Vol.  xix,  Nov., 
1908. 

2  See    also    Floyd    and    Lucas,    Journ.    Med.    Research,    VoL 
xxi,  Sept.,  1909. 


XII.     THE  PRINCIPLES    UNDERLYING  THE 

TREATMENT   OF    SYPHILIS  WITH 

SALVARSAN  ("  606  ") 

As  a  result  of  some  of  his  earliest  researches  and 
in  entire  accordance  with  his  views  as  already  set 
forth  under  "Antitoxins,"  Ehrlich  has  always  held 
that  the  action  of  a  chemical  substance  on  a  given 
cell  denotes  the  existence  of  definite  chemical  affini- 
ties between  the  substance  and  the  cell.  Applying 
this  conception  to  the  germicidal  action  of  chem- 
icals, he  maintains  that  the  latter  must  have  a 
certain  chemical  affinity  for  the  parasites  in  order 
to  kill  them.  Substances  having  such  affinities 
he  terms  "  parasitotropic."  It  is  clear,  however, 
that  substances  which  can  destroy  parasites  will 
also  be  poisonous  for  the  animal  body,  i.e.,  they 
will  have  chemical  affinity  for  the  vital  organs  of 
the  host.  They  are,  therefore,  also  "  organotropic." 
In  the  employment  of  chemical  substances  in  com- 
bating infectious  diseases  it  follows  that  success 
can  only  be  attained  if  their  chemical  affinity  for 
the  infecting  parasite  bears  certain  relations  to  their 

affinity  for  the  infected  body. 

179 


i8o  IMMUNE  SERA 

In  his  researches  on  trypanosomes  Ehrlich  found 
that  if  the  dose  of  germicide  administered  to  an 
infected  animal  was  too  small  to  kill  all  the  para- 
sites, there  developed  after  a  time  a  strain  of  organ- 
isms which  were  resistant  to  the  further  action  of 
the  germicide.  It  was  usually  futile  to  repeat  the 
dose,  for  the  resistant  parasites  would  survive. 
At  the  same  time  the  interesting  observation  was 
made  that  this  resistance  manifested  itself  only 
in  the  animal  body;  in  vitro  the  parasites  could 
still  be  filled  by  the  germicide  in  question.  It  was 
also  found  that  the  various  chemical  substances 
which  exerted  therapeutic  effects  in  animals  infected 
with  trypanosomes  could  be  grouped  into  three 
classes,  namely — i,  various  arsenicals  (arsenious 
acid,  atoxyl,  arsacetin,  arsenophenylglycin,  and 
finally  "606,");  2,  certain  azo  dyes  (among  them 
trypan  red,  trypan  blue,  and  trypan  violet) ;  and  3, 
certain  basic  triphenylmethane  dyes  (among  them 
parafuchsine,  methyl  violet,  pyronine,  etc.).  Against 
each  of  these  classes  it  was  possible  to  produce  speci- 
fically resistant  strains  of  trypanosomes,  so  that  a 
strain  which  had  been  made  resistant  to  fuchsin  was 
also  resistant  to  related  basic  dyes,  but  vulnerable 
to  the  azo  dye  and  to  the  arsenicals.  Moreover, 
by  appropriate  treatment  it  was  found  possible 
to  produce  strains  resistant  against  all  three  classes 
of  trypanocidal  agents. 

According  to  Ehrlich  the  union  of  the  chemical 


TREATMENT  OF  SYPHILIS  WITH  SALVARSAN    181 

with  the  parasites  is  brought  about  by  certain 
"  chemoreceptors  "  of  the  parasites.  The  arsenic 
compounds,  for  example,  are  anchored  by  arseno- 
receptors.  When  a  strain  therefore  becomes  re- 
sistant to  the  arsenicals  one  might  suppose  that 
this  was  due  to  the  parasite  ridding  itself  of  its 
arseno-receptors ;  similarly  also  with  the  other 
classes  of  trypanocidal  substances.  But  this  appears 
not  to  be  the  case,  for,  as  we  have  already  said,  the 
resistance  is  manifested  only  in  the  animal  body, 
and  not  in  vitro.  Ehrlich  explains  this  by  assuming 
that  in  the  resistant  strain  the  affinity  of  that  par- 
ticular chemo-receptor  has  been  reduced,  so  that 
when  the  germicidal  agent  is  introduced  into  an 
animal  infected  with  the  resistant  strain,  the  pro- 
portion of  distribution  of  the  germicide  is  altered 
in  favor  of  the  chemo-receptors  of  the  organism. 
In  other  words  the  organotropic  affinity  is  greater 
than  the  parasitotropic  affinity.  Had  the  parasite's 
chemo-receptors  quite  disappeared  there  should 
have  been  no  difference  in  the  resistance  as  mani- 
fested in  the  animal  and  in  vitro. 

In  searching  for  germicidal  substances  whose 
parasitotropic  affinity  should  be  great  in  comparison 
to  their  organotropic  affinity,  Ehrlich  made  careful 
pharmacological  studies  with  each  of  the  three 
classes  of  trypanocidal  substances  already  mentioned. 
After  testing  homologues  and  substitution  products 
of  almost  every  variety  he  finally  concluded  that 


ig2  IMMUNE  SERA 

dioxydiamidoarsenobenzol  fulfilled  the  require- 
ments. As  each  of  the  substances  was  tested  it 
received  a  laboratory  number  for  identification; 
dioxydiamidoarsenobenzol  bore  the  serial  number 
606,  whence  the  designation  by  which  this  substance 
is  still  commonly  known.  Its  trade  name  is  "  Sal- 
varsan." 

It  is  not  our  purpose,  in  these  pages,  to  enter  into 
the  chemistry  of  "  606,"  or  to  discuss  the  treatment 
of  syphilis  by  this  drug.  Suffice  it  to  say  that 
Ehrlich  lays  considerable  stress  on  the  fact  that 
in  606  the  arsenic  is  in  the  trivalent  unsaturated 
form.  Pentavalent  arsenic  compounds,  he  believes, 
are  less  efficient  in  their  trypanocidal  action.  As 
supplied  in  the  market,  Salvarsan  is  a  bright  yellow 
powder  containing  theoretically  34.15  per  cent 
arsenic.  It  is  the  hydrochloride  of  dioxydiami- 
doarsenobenzol, and  is  administered  by  suspending 
or  dissolving  it  in  water  with  the  addition  of  NaOH 
to  neutralize,  thus  forming  dioxydiamidoarseno- 
benzol plus  NaCl  and  H2O.  Subcutaneous,  intra- 
muscular, and  intravenous  injections  have  been 
employed,  as  also  solutions  and  suspensions  of  the 
drug.  The  results  in  the  treatment  of  syphilis 
have  been  encouraging,  but  the  time  has  not  yet 
come  to  express  a  definite  opinion  concerning  the 
ultimate  value  of  this  drug. 

The  principles  here  outlined,  however,  deserve 
to  be  carefully  studied  and  tested  experimentally, 


TREATMENT  OF  SYPHILIS  WITH  SALVARSAN     183 

for  if  correct  they  point  the  way  for  devising  an 
effective  therapy  for  many  infections  at  present 
quite  beyond  our  control. 

LITERATURE 

EHRLICH-BOLDUAN.  Collected  Studies  in  Immunity, 
(Chapter  XXXIV)  1910.  Wiley  &  Sons,  New 
York. 

EHRLICH.  Experimental  Researches  on  Specific  Thera- 
peutics. 1909.  Hoeber,  New  York. 

EHRLICH  and  HATA.  Die  Experimented  Chemothera- 
pie  der  Spirillosen.  1910.  Springer,  Berlin. 

MARTINDALE  and  WESTCOTT.  "  Salvarsan  "  ("  606  "). 
1911.  Hoeber,  New  York. 

WECHSELMANN.  The  Treatment  of  Syphilis  with  Di- 
oxydiamidoarsenobenzol.  (English  translation  by 
A.  L.  Wolbarst).  1911.  Rebman  Company,  New 
York. 


APPENDIX  A 

THE    WASSERMANN   TEST   FOR    SYPHILIS. 

As  has  already  been  pointed  out  on  page  72, 
Wassermann  applied  the  principle  of  the  Bordet- 
Gengou  phenomenon  to  the  detection  of  syphilis 
antibodies  in  the  serum  and  cerebrospinal  fluid  of 
persons  infected  with  syphilis.  In  the  few  years 
which  have  elapsed  since  Wassermann's  first  pub- 
lication, the  reliability  of  this  method  of  diagnos- 
ing syphilis  has  been  confirmed  by  a  large  number 
of  investigators,  and  it  has  already  proven  of  con- 
siderable value  in  several  departments  of  medicine. 
In  response  to  numerous  requests,  the  writer  has 
undertaken  to  give  a  clear  description  of  the  test, 
together  with  a  brief  review  of  the  results  thus  far 
achieved  by  its  use. 

When  an  animal  is  repeatedly  injected  with  red 
blood  cells  of  another  species,  it  reacts  to  such  in- 
jections by  producing  substances  in  its  serum  which 
have  the  power  to  dissolve  these  foreign  blood  cells. 
Examined  by  means  of  a  test  tube  experiment,  it 
is  found  that  the  serum  exerts  this  power  only 
while  it  is  fresh.  Serum  several  days  old  is  unable 
to  dissolve  the  recj  cells.  The  fresh  serum  also 

'85 


l86  APPENDIX 

loses  its  solvent  power  by  exposure  to  heat,  say  to 
55°  C.  Investigations  showed  that  the  solvent 
action  could  be  restored  to  these  sera  by  the  addi- 
tion of  small  quantities  of  a  fresh  normal  serum, 
i.e.  of  a  serum  which  by  itself  had  no  solvent  power 
whatever.  The  inactive  serum  had  thus  been 
reactivated.  The  original  specific  dissolving  serum 
therefore  contained  two  substances,  one  of  which 
is  very  labile  and  the  other  stable.  The  stable 
substance  is  specific  for  the  blood  cells  against 
which  it  is  directed,  i.e.  against  the  cells  used  for 
immunizing  the  animal.  It  is  called  the  "  immune 
body,"  or  the  "  amboceptor."  The  labile  sub- 
stance, as  we  have  seen,  is  present  in  all  fresh  sera, 
and  is  spoken  of  as  the  " complement."  The  action 
of  the  immune  body  seems  to  consist  in  bringing 
the  solvent  action  of  the  complement  to  bear  on 
the  given  cells.  We  must  conceive  that  the  com- 
plement possesses  the  solvent  power,  but  has  no 
way  of  laying  hold  of  the  cell  to  be  dissolved.  The 
immune  body  merely  effects  this  combination. 
Ehrlich's  diagram  on  page  66  will  serve  to  make 
this  conception  clear. 

All  that  has  been  said  regarding  immune  bodies 
and  complement  for  the  solution  of  blood  cells, 
holds  for  the  substances  which  effect  destruction  of 
bacteria  when  bacteria  are  used  for  immunization. 
In  fact,  the  process  is  the  same,  no  matter  what 
cells  are  injected  into  the  animal.  The  immune 
body  is  always  directed  specifically  against  the 
cells  injected,  and  against  no  others. 


APPENDIX 

As  can  be  seen  from  Ehrlich's  diagrams,  the 
bacteria  or  blood  cells  combine  directly  only  with 
the  immune  body.  The  complement,  as  already 
said,  has  no  way  of  laying  hold  of  the  cells.  As 
soon  as  the  bacteria  or  cells  have  anchored  the 
immune  body,  however,  conditions  change.  The 
combination  at  once  attracts  and  unites  with  the 
complement.  If  the  amount  of  complement  is  not 
too  large,  the  combination  may  unite  with  all  of  it, 
i.e.  may  abstract  the  complement  from  the  serum. 

Just  let  us  examine  this  by  means  of  an  illustra- 
tion: Let  us  suppose  we  have  immunized  an  ani- 
mal with  typhoid  bacilli,  and  have  obtained  a 
specific  serum  directed  against  these  bacilli.  This 
serum  has  been  inactivated  by  heating  it  to  55°  C., 
so  that  now  it  will  act  on  typhoid  bacilli  only  when 
some  fresh  normal  serum  is  added  to  complement 
the  immune  body.  For  this  purpose  we  have 
provided  ourselves  with  some  freshly  drawn  serum 
from  a  guinea  pig.  The  guinea  pig  serum,  there- 
fore, is  the  "complement."  On  mixing  typhoid 
bacilli  with  the  specific  immune  serum  and  then 
with  the  complement,  these  three  factors  enter  into 
combination,  and  this  results  in  the  destruction  of 
the  typhoid  bacilli.  The  quantities  can  easily  be 
so  arranged  that  this  combination  uses  up  all  of  the 
complement,  so  that  the  fluid  contains  not  a  trace 
of  free  complement  after  the  substances  have  com- 
bined. 

Suppose,  now,  that  we  also  had  a  specific  serum 
obtained  by  injecting  an  animal  with  red  blood 


188  APPENDIX 

cells,  for  example,  by  injecting  a  rabbit  with  sheep 
blood  cells.  This  rabbit  serum  would  then  be 
specifically  directed  against  sheep  blood  cells.  Let 
us  inactivate  this  serum,  by  heating  it  to  55°  C.,  so 
that  now  it  requires  the  addition  of  a  fresh  normal 
serum  to  exert  its  solvent  effect.  For  this  purpose 
we  can  again  use  fresh,  normal  guinea-pig  serum. 
When,  then,  we  mix  sheep  blood  cells  with  our 
specific  immune  serum  (against  sheep  blood  cells) 
and  with  the  complement,  i.e.,  with  fresh  normal 
guinea-pig  serum,  all  three  factors  unite,  and  bring 
about  destruction  of  the  red  blood  cells.  This  is 
manifested  by  the  blood  cells  dissolving  and 
giving  off  their  haemoglobin  to  the  rest  of  the 
fluid. 

Let  us  now  suppose  we  have  carried  out  the  first 
part  of  this  experiment,  that  with  the  typhoid  bacilli, 
and  have  left  typhoid  bacilli,  specific  typhoid  serum 
and  complement  in  contact  for  several  hours  in  a 
warm  place  in  order  to  cause  the  three  factors  to 
combine.  At  the  end  of  this  time  let  us  add  sheep 
blood  cells  and  the  specific  serum  directed  against 
sheep  cells,  but  let  us  add  no  further  complement, 
because  the  fresh  guinea-pig  serum  was  able,  as  we 
saw,  to  serve  as  as  complement  also  for  the  blood 
combination.  The  mixture  is  again  placed  in  a 
warm  place  for  several  hours,  and  then  for  twenty- 
four  hours  in  the  refrigerator,  after  which  it  is  ex- 
amined. We  shall  find  that  no  haemolysis  has 
occurred,  from  which  we  conclude  that  the  previous 
combination  (typhoid  bacilli,  immune  serum  and 


APPENDIX  189 

complement),  had  used  up  all  the  complement,  and 
left  none  for  the  blood  combination. 

If  we  were  to  repeat  the  whole  experiment,  but 
leave  out,  in  the  first  part  of  the  test,  say  the  spe- 
cific typhoid  serum,  we  should  find  that  the-  blood 
cells  would  be  dissolved.  This  is  readily  under- 
stood when  it  is  remembered  that  then  we  would 
have  only  typhoid  bacilli  and  complement,  two 
factors  which  cannot  ccmbine  directly.  The  com- 
plement would  therefore  be  left  free  to  act  in  the 
blood  combination. 

If  hasmolysis  occurs  we  may  therefore  conclude 
that  one  of  the  factors  in  the  first  combination  was 
absent,  and  conversely,  if  hasmolysis  does  not  occur, 
we  know  that  the  first  combination  must  have 
been  perfect,  i.e.  all  three  factors  must  have  been 
present. 

It  is  at  once  apparent  that  in  adapting  this  test 
to  the  detection  of  syphilis  antibodies,  pure  cultures 
of  the  causative  organism,  i.e.  of  the  "antigen," 
were  not  available.  Wassermann  therefore  made 
use  of  extracts  of  syphilitic  organs  rich  in  spiro- 
chastes  in  place  of  the  typhoid  bacilli,  and  used 
either  the  serum  or  the  cerebrospinal  fluid  of  the 
suspected  case  in  place  of  the  typhoid  antiserum. 
The  rest  of  the  test  was  similar  to  that  described 
above.  When  haemolysis  of  the  sheep  cells  occured, 
Wassermann  said  it  showed  that  the  first  combina- 
tion was  incomplete;  when  haemolysis  was  com- 
pletely inhibited,  it  showed,  he  said,  that  the  first 
combination  was  perfect,  i.e.  that  the  serum  or 


190  APPENDIX 

spinal  fluid  contained  syphilis  antibody.  As  in 
most  such  tests,  only  a  positive  result  determines ; 
a  negative  result  does  not  necessarily  exclude  the 
presence  of  syphilitic  infection. 

While  the  above  exposition  will  serve  to  fix  the 
general  plan  of  the  test  in  the  mind  of  the  reader, 
we  must  at  once  say  that  the  mode  of  action  is  not 
as  simple  as  Wassermann  first  believed.  Before 
going  into  this  phase  of  the  subject,  it  will  be 
advisable  to  present  a  description  of  the  technique 
of  the  test. 

For  carrying  out  the  test  the  following  materials 
are  required: 

(i)"  Antigen,"  i.e.  fluid  containing  syphilis  ma- 
terial. This  is  comparable  to  the  pure  culture  of 
typhoid  in  the  test  described  above. 

(2)  Serum  or  cerebrospinal  fluid  from  the  patient 
to  be  examined. 

(3)  Sheep  blood  cells. 

(4)  Haemolytic  antibody,  i.e.    inactivated  serum 
of  a  rabbit  immunized  against  sheep  blood  cells. 

(5)  Complement,  i.e.  fresh  normal  serum  from  a 
guinea-pig. 

For  the  syphilis  antigen  it  is  best  to  use  the 
organs  of  a  syphilitic  foetus,  i.e.  one  dead  of  heredi- 
tary syphilis,  as  these  tissues  are  particularly  rich 
in  spirochaetes.  The  organs  are  chopped  up  and 
macerated  in  a  clean  vessel  in  a  mixture  composed 
of  water,  1000;  NaCl,  8.5;  carbolic  acid,  5.0;  one 
part  of  the  tissue  to  four  of  the  fluid.  The  mixture 
is  shaken  in  a  shaking  apparatus  for  twenty  hours ; 


APPENDIX 

the  supernatant  fluid  poured  off  and  centrifuged  so 
as  to  be  perfectly  clear.* 

The  serum  for  the  test  is  collected  from  the 
patient  in  the  usual  way  by  drawing  from  5  to 
10  cc.  of  blood  from  a  vein  at  the  elbow,  placing 
the  blood  in  a  sterile  test  tube  and  allowing  it  to 
clot.  Cerebrospinal  fluid,  obtained  by  lumbar 
puncture,  is  preserved  with  0.5%  carbolic  acid,  and 
then  strongly  centrifuged  so  as  to  make  it  perfectly 
clear. 

The  sheep  blood  cells  are  obtained  by  defibrinat- 
ing  sheep  blood,  centrifuging  and  washing  the  blood 
cells  repeatedly  with  normal  salt  solution  to  remove 
traces  of  adherent  serum.  A  5%  suspension  in  salt 
solution  is  used. 

The  haemolytic  antibody  consists  of  the  serum  of 
a  highly  immunized  (against  sheep  blood  cells) 
rabbit,  the  serum  being  inactivated  by  heating  to 
56°  C.  In  the  tests  cited  by  Wassermann,  one  cc. 
of  a  1/1500  dilution  of  serum  dissolved  one  cc.  of 
5%  suspension  of  sheep  blood  cells  at  37°  C.  in 
two  hours. 

The  complement  consists  of  freshly  drawn  guinea- 
pig  serum.  The  test  is  carried  out  as  follows : 

To  constant  quantities  of  spinal  fluid  (e.g.  i  cc. 
of  the  i/io  dilution)  decreasing  amounts  of  the 
extract  of  organs  are  added,  thus  0.2,  o.i,  0.05  cc. 
Then  i  cc.  of  a  i/io  dilution  of  fresh  normal  guinea- 

*  In  a  very  recent  article,  Wassermann  states  that  more  uni- 
formly active  extracts  can  be  obtained  by  using  96%  alcohol  in 
place  of  the  water  in  the  above  formula. 


1.9  2  APPENDIX 

pig  serum  is  added,  and  the  mixtures  allowed  to 
remain  in  contact  at  37°  C.  for  one  hour  in  order  to 
bind  the  complement. 

In  this  mixture  we  have  antigen;  we  may  or  may 
not  have  antibody;  we  have  complement. 

If  the  antibody  is  present,  the  complement  will 
be  anchored  by  the  combination,  and  so  be  unavail- 
able for  the  haemolytic  test  next  in  order.  If  no 
antibody  is  present,  the  complement  will  still  be 
free  to  act  in  the  haemolytic  test. 

At  the  end  of  the  hour,  we  add  to  the  above 
mixtures:  one  cc.  of  a  5%  suspension  of  sheep 
blood  cells,  and  one  cc.  of  the  amboceptor  dilution 
containing  double  the  solvent  dose  for  that  amount 
of  sheep  blood  cells.  Thus,  if  the  titer  of  the 
haemolytic  serum  is  1/1800,  we  take  one  cc.  of  a 
dilution  1/900. 

All  the  tubes  are  made  up  to  the  same  volume 
with  normal  salt  solution,  namely,  to  5  cc.,and  are 
then  placed  in  the  incubator  at  37°  C.  and  kept 
there  for  two  hours.  Then  they  are  placed  on  ice 
until  the  next  day,  when  the  results  are  noted- 
The  whole  procedure  is  clearly  shown  by  the  pro- 
tocol from  Wassermann  and  Plaut  reproduced  on 
page  193. 

Few  experiments  in  immunity  require  such  care- 
ful technique,  or  are  open  to  so  many  sources  of 
error  as  this  serum  test  for  syphilis.  In  view, 
too,  of  the  enormous  responsibility  assumed  in 
making  a  positive  diagnosis  of  syphilis,  it  is  appa- 
rent that  the  test  should  only  be  undertaken  by 


APPENDIX 


193 


J 

Haemoly- 

tic  Ambo- 

Syphilit. 
Foetus 
Extract. 
(0.2  gm.) 
One  c.c. 
of  I  Dilu- 
tion. 

Spinal  Fluid 
of  Patient 
M.  0.2,  i.e.. 
One  c.c.  of 
the  £  Dilu- 
tion. 

Normal 
Guinea- 
pig  Serum 

O.I    CC., 

i.e.  i  cc. 
of  a  A 
Dilution. 

ceptor 

I  CC. 

equals 
Double 
the 
Solvent 
dose  for 

Sheep 
Blood 
Cells  i  cc. 
of  a  5% 
Suspen- 
sion. 

Results. 

i  cc.  of  a 

5%  Sus- 

pension. 

0.2 

0.2 

I.O 

I.O 

i  .0 

Complete  inhibition 

of  haemolysis 

O.I 

0.2 

I.O 

I.O 

I.O 

Compl.  inhibition 

0.2 

0.  I 

I.O 

I.O 

i  .0 

Marked  inhibition 

O.I 

O.  I 

I.O 

I.O 

I.O 

Marked  inhibition 

0.2 



I.O 

I.O 

I.O 

Complete  solution 

O.I 



I.O 

I.O 

I.O 

Complete  solution 



0.2 

I.O 

I.O 

i  .0 

Complete  solution 



O.I 

I.O 

I.O 

I.O 

Complete  solution 

Spinal  Fluid 

of 

Non-syphil. 

Person. 

0.2 

0.  2 

1  .0 

I.O 

I.O 

Complete  solution 

0.  I 

0.  2 

I.O 

I.O 

I.O 

Complete  solution 

O.  2 

•    0.  I 

I.O 

I.O 

I.O 

Complete  solution 

O.I 

O.I 

1  .0 

1  .0 

I.O 

Complete  solution 



0.2 

I.O 

I.O 

I.O 

Complete  solution 

O.  I 

I.O 

I.O 

I.O 

Complete  solution 

highly  trained  laboratory  workers.  On  the  other 
hand,  most  who  have  busied  themselves  with  the 
test  agree  that  suitable  controls  always  lead  to  a 
detection  of  possible  sources  of  error,  and  that 


IQ4  APPENDIX 

therefore  the  reaction,  when  properly  performed, 
can  be  relied  upon. 

When  the  test  was  first  published  Wassermann 
regarded  the  reaction  which  occurred  as  one  between 
mutually  specific  bodies,  i.e.  between  antigen  and 
antibody,  the  resulting  combination  having  the 
power  to  anchor  the  complement.  Through  the 
work  of  Marie  and  Levaditi,  of  Landsteiner,  Miiller 
and  Potzl,  of  Weil  and  Braun,  and  still  other  in- 
vestigators, it  soon  became  apparent  that  the  test 
could  also  be  carried  out  by  using  extracts  of  non- 
syphilitic  tissue,  i.e.  of  other  pathological  tissue  or 
normal  tissue.  That,  of  course,  meant  that  the 
view  of  a  reciprocal  specific  relation  between  anti- 
body and  organ  extract,  in  the  sense  that  typhoid 
antibody  and  typhoid  bacilli  are  reciprocally  re- 
lated, had  to  be  abandoned.  This  does  not,  how- 
ever, effect  the  reliability  of  the  reaction  for  diag- 
nostic purposes,  for  it  has  been  found  that  positive 
results  are  still  only  obtained  when  the  serum  or 
spinal  fluid  is  of  syphilitic  origin.* 

Working  under  Wassermann's  direction,  Forges 
and  Meier  studied  the  nature  of  the  substances 
concerned  in  the  reaction,  and  began  by  precipi- 
tating the  organ  extracts  with  alcohol  and  testing 

*It  may  be  well  to  state  that  according  to  Landsteiner,  Muller 
and  Potzl  the  serum  of  animals  infected  with  dourine  (trypanoso- 
miasis)  also  gives  rise  to  inhibition  of  haemolysis  when  tested 
according  to  the  above  method.  This  has  been  confirmed  by 
Hartoch  and  Yakimoff.  Whether  this  will  affect  the  value  of  the 
Wassermann  test  in  humans  can  only  be  decided  by  further 
clinical  tests,  especially  in  cases  of  human  trypanosomiasis. 


APPENDIX  195 

the  resulting  precipitate  and  clear  fluid  separately. 
It  was  found  that  the  substance  concerned  in  the 
reaction  was  soluble  in  alcohol,  and  the  authors 
thereupon  made  alcoholic  extracts  of  the  syphilitic 
organs.  These  worked  satisfactorily  in  making  the 
test.  It  was  natural  to  think  that  the  substance 
which  effected  the  reaction  might  be  related  to  the 
lipoids,  and  so  the  authors  next  studied  the  be- 
havior of  alcoholic  extracts  of  normal  human  and 
animal  organs.  While  these  extracts  also  sufficed 
to  produce  the  reaction,  the  authors  felt  that  they 
were  not  as  active  as  extracts  from  syphilitic 
organs.  After  it  had  been  found  that  alcoholic  ex- 
tracts could  be  used  for  the  test,  a  number  of 
authors  almost  simultaneously  published  favorable 
results  with  chemically  defined  substances.  Forges 
and  Meier  used  lecithin,  Levaditi  glycocholate  of 
soda,  Sachs  and  Altmann  oleate  of  soda,  and 
Fleischmann  even  used  vaseline.  The  last-named 
also  used  cholesterin  with  favorable  results,  but 
Forges  and  Meier  obtained  only  negative  results 
with  this  substance.*  On  the  whole,  however,  it 
seems  that  the  extracts,  especially  of  syphilitic 
organs,  give  the  most  uniform  results. 

At  the  present  time,  therefore,  Wassermann  be- 
lieves that  the  really  active  principle  in  the  antigen 
may  be  a  combination  of  lipoids  with  certain  protein- 
like  substances,  and  that  the  latter  component, 

*  See  especially  Noguchi,  The  Relation  of  Protein,  Lipoids  and 
Salts  to  the  Wassermann  Reaction,  Journ  Exp  Medicine,  vol. 
xi,  1909. 


196  APPENDIX 

when  it  is  derived  from  syphilitic  material,  has 
something  of  a  specific  character.  In  this  connec- 
tion Wassermann  refers  to  the  researches  of 
Noguchi,  Landsteiner,  and  others  which  show  that 
minute  quantities  of  proteid  mixed  with  lipoids 
may  cause,  extensive  alterations  in  the  physico- 
chemical  behavior  of  the  latter.  He  thinks  that 
under  certain  circumstances  this  proteid  component 
may  play  an  important  role  in  determining  the 
reliability  of  the  reaction,  a  view  which  is  borne  out 
by  the  investigations  of  Neisser  and  Bruck. 

While  Forges  and  Meier  were  engaged  in  the 
studies  just  mentioned,  Fornet  and  also  Michaelis 
showed  that  when  the  serum  of  individuals  infected 
with  syphilis  was  mixed  with  certain  antigens  a 
zone  of  precipitation  might  at  times  be  observed  at 
the  point  of  contact  of  the  two  fluids.  The  antigen 
employed  by  Fornet  was  serum  from  individuals  in 
the  florid  stage  of  syphilis ;  Michaelis  used  extracts 
of  organs  from  a  syphilitic  foetus.  This  of  course 
agrees  with  what  was  already  known  from  the  work 
of  Bordet,  Gengou,  and  Gay.  In  fact,  according 
to  Gay,  the  deflection  or  absorption  of  complement, 
on  which  the  Bordet-Gengou  test  depends,  may  be 
due  to  the  precipitate  formed  in  the  combination. 

Forges  and  Meier  thereupon  tested  the  alcoholic 
extracts,  and  solutions  of  lecithin  and  of  glycocho- 
late  of  soda  to  see  whether  this  zone  of  precipita- 
tion was  at  all  constant,  and  whether  it  might  not 
be  possible  to  substitute  such  a  simple  precipitation 
test  for  the  complicated  Wassermann  reaction. 


APPENDIX  197 

While  it  was  found  that  the  test  was  roughly  spe- 
cific, it  was  soon  realized  that  a  precipitate  might 
at  times  be  produced  with  the  serum  of  surely 
non-syphilitic  individuals,  and  similar  unfavorable 
results,  have  since  been  published  by  other  authors. 
At  the  present  time,  therefore,  the  only  reliable 
serum  diagnosis  of  syphilis  is  that  based  on  the 
absorption  of  complement. 

The  results  obtained  with  the  Wassermann  test  are 
well  reflected  in  the  findings  of  Fleischmann,  as 
follows : 

The  total  number  of  persons  tested  was  230,  of 
which  38  were  controls.  None  of  the  latter  gave  a 
positive  reaction.  The  other  cases  can  be  arranged 
into  four  groups  thus : 

1 i )  Cases  surely  syphilitic,  with  clinically  manifest 
signs  of  syphilis  at  the  time  of  the  test.   Of  89  such 
cases  tested,  83  gave  a   positive  reaction  (93%). 

(2)  Cases    surely  syphilitic   but   without  clinical 
symptoms   at    the  time   of  the  test.     Of  64  such 
cases  tested,  33  gave  a  positive  reaction  (52%),  and 
31  gave  a  negative  reaction  (48%). 

(3)  Cases  with  symptoms  suggestive  of  syphilis, 
and  with  an  indefinite  history  of  infection.     Of  32 
such  cases,  16  gave  a  positive  reaction  (50%),  and 
the  rest  a  negative  reaction. 

(4)  Surely  syphilitic  individuals  showing  cutaneous 
lesions  which  the  dermatologists  diagnosed  as  very 
probably  not  syphilitic.     Of  7  such  cases,  i  gave  a 
positive  reaction  and  the  rest  a  negative  reaction. 

Bruck  and  Stern  tested  378  cases  suspected  to  be 


198  APPENDIX 

syphilitic,  and  obtained  a  positive  reaction  in  204. 
They  also  tested  157  surely  non-syphilitic  individuals 
as  controls,  and  found  all  but  two  negative.  These 
two  gave  a  doubtful  reaction. 

In  a  recent  paper  Wassermann  has  collected  data 
on  about  3000  tests,  as  follows:  There  were  1010 
tests  on  cases  surely  non-syphilitic  (controls),  and 
not  one  of  these  gave  a  positive  reaction.  Of  the 
1982  surely  syphilitic  cases  tested,  those  examined 
at  the  time  when  they  had  manifest  symptoms 
reacted  in  about  90%  of  the  cases.  When  the  cases 
tested  were  without  manifest  symptoms,  so-called 
"latent  syphilitics,"  about  50%  reacted. 

As  a  matter  of  interest  it  may  be  mentioned  that 
Blumenthal  and  Wile  tested  the  urine  of  syphilitic 
individuals,  and  found  that  this  too  would  give  the 
reaction. 

Marie  and  Levaditi  examined  the  cerebrospinal 
fluid  of  30  cases  of  general  paresis.  All  but  two  of 
the  cases  gave  a  positive  reaction.  When  the  serum 
was  tested  in  place  of  the  cerebrospinal  fluid,  the 
percentage  of  positive  findings  dropped  to  59%. 

Michaelis  examined  20  cases  of  general  paresis 
and  obtained  a  positive  reaction  in  1 8  of  them. 

Citron  examined  43  tabetics  and  paretics,  and  ob- 
tained a  positive  reaction  in  34  cases  (79%).  He 
also  tested  the  serum  of  108  persons  surely  infected 
with  syphilis,  or  suspected  to  be  infected,  and  ob- 
tained a  positive  reaction  in  80  (74%).  None  of  the 
sera  from  156  surely  non-syphilitic  individuals  gave 
a  positive  reaction. 


APPENDIX 


199 


Favorable  reports  have  also  been  published  con- 
cerning the  reliability  of  the  test  in  ophthalmology, 
dermatology,  and  other  departments  of  medicine. 

From  the  researches  of  Noguchi  and  others  it 
appears  that  with  the  progress  of  the  cure  of  syphi- 
litic infection  not  only  do  the  symptoms  of  the 
disease  abate,  but  the  blood  reaction  also  grows 
weaker  and  weaker,  until  ultimately,  when  cure 
has  been  established,  the  reaction  can  no  longer 
be  obtained.  This  is  of  clinical  interest,  for 
it  enables  one,  by  means  of  the  serum  test,  to 
control  the  duration  and  efficacy  of  anti-syphilitic 
treatment.  At  the  same  time  it  must  be  remem- 
bered that  the  disappearance  of  the  reaction  need 
not  necessarily  be  permanent.  Its  reappearance,  of 
course,  signifies  that  the  virus  is  still  present  in  the 
body. 


APPENDIX  B 

NOGUCHI'S     MODIFIED     WASSERMANN     REACTION.* 

A  MODIFICATION  of  the  Wassermann  test  recently 
devised  by  Noguchi  has  been  found  both  simple 
and  reliable.  In  principle,  the  test  is  the  same  as 
Wassermann's,  the  only  difference  being  in  the 
hasmolytic  system  employed.  By  making  use  of  an 
antihuman  amboceptor,  Noguchi  avoids  having  con- 
stantly to  obtain  fresh  sheep  blood  cells.  In  addi- 
tion there  is  some  gain  in  accuracy  owing  to  the 
fact  that  human  blood  serum,  in  the  quantities 
ordinarily  employed  in  the  Wassermann  test,  often 
contains  normal  amboceptors  for  sheep  blood  which 
interfere  with  the  reaction. 

The  reagents  employed  by  Noguchi  are  as  follows : 

1.  Antihuman  haemolytic  amboceptor.     This  con- 
sists of  a  serum  obtained  from  rabbits  immunized 
with  washed  human  erythrocytes. 

2.  Complement.     This  consists   of  fresh  guinea- 
pig  serum. 

3.  "  Antigen."     Alcoholic  extracts  of  organs,  or 
crude  preparations  of  lecithin.     The  preparation  of 
the  organ  extracts  has  been  described  on  page  152. 

*  Noguchi,  A  New  and  Simple  Method  for  the  Serum  Diagonosis 
of  Syphilis,  Journ.  Exp.  Medicine,  Vol.  XI,  1909. 

200 


APPENDIX  201 

In  using  lecithin,  0.3  gms.  lecithin  are  dissolved  in 
50  cc.  absolute  alcohol,  shaken  with  50  cc.  physio- 
logical salt  solution  and  filtered.  The  filtrate  must 
be  clear. 

4.  Suspension  of  human  blood  corpuscles.     One 
drop  of  blood  from  a  normal  individual  is  mixed 
with  4  cc.  physiological  salt  solution. 

5.  Serum  to  be  tested.     About  10  or  15  drops  of 
blood  are  collected  in  a  small  tube  and  allowed  to 
clot.     The  clear  serum  is  used  for  the  tests. 

Method. — Take  six  clean  test  tubes,  size  10  cm. 
by  i  cm.  Into  the  first  two  of  these  place  one  drop 
from  a  capillary  pipette  of  the  patient's  serum  to  be 
tested.  Into  each  of  the  second  two  tubes  (which 
serve  as  controls)  put  one  drop  of  serum  of  a  syphi- 
litic case  known  to  give  a  positive  reaction.  Into 
each  of  the  third  pair  of  tubes  put  one  drop  of 
serum  of  a  normal  person.  Now  to  each  of  the  six 
tubes  add  i  cc.  of  the  suspension  of  human  blood 
corpuscles  and  0.04  cc.  fresh  guinea-pig  serum  as 
complement.  Lastly,  into  one  tube  of  each  pair 
put  one  drop  of  the  "  antigen  "  solution  from  a 
capillary  pipette.  The  second  tube  of  each  pair 
receives  no  antigen. 

After  being  well  mixed  by  shaking,  the  six  tubes 
are  incubated  at  3  7°  C.  for  one  hour,  after  which  each 
tube  receives  two  units  *  of  antihuman  amboceptor. 
The  tubes  are  returned  to  the  incubator  for  two 

*  The  term  "amboceptor  unit"  is  used  to  designate  the  amount  of 
ambcceptor  which,  on  the  addition  of  the  optimum  amount  of  comple- 
ment, just  suffices  to  produce  complete  haemolysis. 


202 


APPENDIX 


hc'irs  longer,  and  then  kept  at  room  temperature, 
the  reaction  being  read  from  time  to  time.  The 
interpretation  of  the  result  is  identical  with  that  of 
the  Wassermann  reaction. 

The   following   scheme   accompanying   Noguchi's 
article  will  serve  to  elucidate  the  technique. 


First  Central  Set. 

Second  Control  Set. 

Test  for  Diagnosis. 

Test  with  a  known 
svphilitic  -erum. 

Test  with  a  normal 
serum. 

(Positive  reaction.) 

(Negative  reaction.) 

a.  Patient's 

a1  '.   Posi- 

a". Normal 

serum 

tive 

serum 

luetic 

Control  tube 

serum 

without 

Q     b.  Human 

Q     b.  Human 

Q     b.  Human 

"antigen" 

blood 

blood 

blood 

in  each  test 

suspen- 

suspen- 

suspen- 

sion 

sion 

sion 

c.   Comple- 

c.  Comple- 

c.  Comple- 

ment 

ment 

ment 

Determina- 

a.   1 

a'.    I 

a"'l 

tive  tube, 

^     &.     [  Ditto 

^     b.      \  Ditto 

r^     b.     i  Ditto 

contains 

U     c.    J 

U     c.     J 

U     c.    J 

"antigen" 

d.  Antigen 

d.    Antigen 

d.    Antigen 

Incubation  at  37°  C.  for  one  hour. 

Antihuman  haemolytic  amboceptor  to  all  tubes. 

Incubation  at  37°  C.  for  two    hours   longer,  then   at 

room  temp. 

Noguchi  has  very  cleverly  adapted  his  method 
for  clinical  purposes  by  drying  the  various  reagents 
on  paper  and  standardizing  them.  He  prepares 
three  kinds  of  slips,  as  follows: 


APPENDIX  203 

Antihuman  amboceptor  slips,  each  containing  two 
amboceptor  units. 

Complement  slips,  each  containing  sufficient  com- 
plement for  one  tube. 

Antigen  slips,  each  containing  sufficient  for  one 
tube. 

These  papers  can  be  kept  indefinitely  at  room 
temperature  in  a  dry  place.  Prior  to  using  the  slips 
for  a  blood  test  it  is  necessary  to  make  preliminary 
tests  to  prove  their  activity  and  strength. 

In  employing  the  filter  paper  slips  they  are 
dropped  by  means  of  a  forceps  into  the  test  tubes 
containing  the  human  blood  suspension  and  patient's 
serum  in  the  order  and  at  the  intervals  already 
stated  for  the  respective  reagents.  If  an  incubator 
is  not  at  hand,  the  tubes  can  be  kept  warm  by 
carrying  them  in  the  vest  pocket. 


APPENDIX    C 

BLOOD  EXAMINATION  PREPARATORY  TO  TRANSFUSION 

Reasons  for  Making  the  Examination. — We  have 
already  called  attention  to  the  occurrence  of  iso- 
agglutinins,  isohaemolysins,  and  isoprecipitins  and 
their  bearing  on  homologous  transfusion.  The 
mere  occurrence  of  these  substances  in  blood  serum, 
to  be  sure,  does  not  at  all  prove  that  isoagglutina- 
tion  or  isohaemolysis  or  isoprecipitation  occur  when 
such  transfusions  are  done.  In  fact  we  do  not  even 
know  whether  these  substances  exist  at  all  in  the 
blood  plasma.  Nevertheless,  until  we  learn  other- 
wise, it  will  be  well  to  bear  in  mind  the  possible 
danger  from  this  source,  and  to  undertake  no 
transfusions  in  which  examination  shows  the  exist- 
ence of  homologous  antibodies. 

Technique  of  the  Tests. — It  is  evident  that  our 
tests  must  be  reciprocal,  i.e.,  we  must  test  the 
serum  of  both  donor  and  recipient  against  the 
blood  corpuscles  of  the  other.  To  do  this  we  col- 
lect part  of  the  blood  from  each  individual,  part 

in  citrated  salt  solution  and  part  in  a  plain  test- 

204 


APPENDIX  205 

tube.  The  latter  is  allowed  to  clot  and  furnishes 
the  serum;  the  former  is  prevented  from  clotting 
by  the  sodium  citrate  and  serves  to  supply  the 
blood  corpuscles.  Instead  of  using  sodium  citrate, 
Crile,1  defibrinates  the  blood  by  shaking  it  in  a 
test-tube  with  a  glass  bead,  and  suspending  the 
blood  corpuscles  in  physiological  salt  solution. 
Either  method  may  be  used,  though  with  the 
sodium  citrate  it  is  necessary  to  centrifuge,  wash 
the  blood  corpuscles,  and  then  resuspend  them  in 
salt  solution.  The  suspensions  are  usually  5% 
strength. 

In  carrying  out  the  test  equal  parts  of  serum  and 
blood  suspension  are  mixed  in  a  small  test-tube,  or, 
as  Epstein 2  has  suggested,  in  small  pipettes  such 
as  Wright  uses  for  his  opsonic  tests.  After  mixing, 
the  tubes  are  placed  in  the  thermostat  for  two  hours. 
At  the  end  of  this  time  most  of  the  cells  have 
usually  settled  to  the  bottom  and  pronounced 
haemolysis  can  be  seen.  For  finer  grades  of  haemo- 
lysis it  is  usually  necessary  to  allow  the  tubes  to 
stand  over  night  in  the  refrigerator. 

Agglutination,  when  it  occurs,  is  rather  prompt, 
and  can  be  readily  observed  in  the  gross  by  the 
clumping  and  sedimentation  of  the  blood  corpuscles. 

1  Crile,  Hemorrhage    and    Transfusion,    1909,  Appleton  and 
Co.,  New  York. 

2  Epstein    and    Ottenburg,    Archives    of    Internal   Medecine, 
Vol.  iii,  page  286,  1909. 


206  APPENDIX 

It  is  important  in  the  haemolytic  tests  that  all 
the  glassware  be  absolutely  clean  and  dry,  though 
it  need  not  be  sterile. 

In  testing  for  the  presence  of  isoprecipitins,  equal 
parts  of  the  two  sera  are  mixed  in  a  small  test-tube, 
the  mixtures  kept  in  the  incubator  for  two  hours 
and  then  examined. 


APPENDIX   D 

OTHER   REACTIONS 

The  Conglutination  Reaction 

IN  1906  Bordet  and  Gay  described  the  presence 
in  bovine  serum  of  a  substance  having  the  property 
of  producing  a  characteristic  clumping  of  red  blood- 
cells  and  of  accelerating  their  lysis  provided  the 
cells  had  first  been  treated  with  both  sensitizer 
(amboceptor)  and  alexin  (complement) ;  its  action 
was  possible  under  no  other  circumstances.  Subse- 
quently it  was  shown  that  the  same  phenomenon 
could  be  produced  with  bacteria  treated  with  a 
specific  sensitizer  and  alexin.  This  substance  is 
spoken  of  as  "conglutinin."  From  the  work  of 
Bordet,  Streng,  Gay,  and  others  it  would  appear 
that  the  conglutination  reaction  can  be  employed 
to  discover  the  presence  of  specific  sensitizers 
(amboceptors) ,  and  thus  be  applied  to  the  diagnosis 
of  bacterial  infections.  The  experimental  work 
thus  far  done  on  the  subject  is  still  too  meagre  to 
warrant  any  definite  statements  as  to  the  diag- 
nostic value  of  the  reaction.  In  some  infections 
studied  by  Gay  the  conglutination  reaction  appeared 

earlier  than  the  agglutination  reaction. 

207 


20$  APPENDIX 

The  Meiostagmin  Reaction 

Weichardt  called  attention  to  the  fact  that  the 
union  of  antigen  with  its  antibody  in  certain  dilu- 
tions caused  an  increase  in  the  rate  of  diffusion, 
i.e.,  gave  rise  to  changes  in  the  osmotic  pressure 
and  of  the  surface  tension.  Ascoli  showed  that  the 
decrease  in  the  surface  tension  arising  when  bac- 
terial substances  combined  with  their  specific  antigen 
could  be  measured  by  counting  the  number  of  drops 
per  given  time  interval  delivered  from  a  Traube 
stalagmometer.  Thus  where  a  mixture  of  normal 
serum  with  extract  of  typhoid  bacilli  showed  56 
drops,  a  similar  mixture  of  serum  from  a  typhoid 
fever  patient  with  the  extract  showed  58  drops. 
Attempts  have  been  made  to  utilize  the  meiostag- 
min  reaction  in  the  diagnosis  of  various  infectious 
diseases,  and  while  the  results  on  the  whole  have 
shown  the  correctness  of  the  underlying  principles, 
they  have  also  demonstrated  that  other  reactions 
are  far  more  convenient  and  decisive. 

The  Much-Holzmann  Cobra  Venom  Reaction 

It  has  long  been  known  that  cobra  venom  haemo- 
lyzes  red  blood  corpuscles,  and  that  certain  cor- 
puscles, such  as  those  of  man,  dog,  pig,  horse, 
rabbit,  and  guinea-pig  haemolyze  directly  on  mixing 
them  with  cobra  v-enom,  while  others  require  the 


APPENDIX  209 

intervention  of  an  activating  substance.  To  the 
latter  class  belong  the  blood  corpuscles  of  ox, 
sheep,  and  goat.  As  already  pointed  out  in  dis- 
cussing snake  venoms,  the  activating  substance  is 
present  in  blood  serum;  it  is  also  present  in  com- 
mercial lecithin.  Haemolysis  of  either  group  of 
blood  corpuscles  can  be  inhibited  by  means  of 
cholesterin,  though  just  how  this  substance  acts  is 
not  clear.  Much  and  Holzmann  showed  that  the 
blood  serum  of  patients  suffering  from  various 
mental  disorders,  especially  dementia  praecox,  and 
manic-depressive  insanity  frequently  inhibits  hae- 
molysis of  human  red  blood  corpuscles,  and  they 
suggested  that  the  reaction  could  be  used  for 
diagnostic  purposes.  While  it  appears  to  be  true 
that  the  psychoses  yield  the  largest  proportion  of 
positive  reactions,  the  value  of  the  reaction  for 
diagnostic  purposes  is  practically  nil.  At  the  same 
time  it  is  interesting  to  note  that  diseases  of  the 
nervous  system  accompanied  by  demonstrable 
lesions  of  the  nerve  tissue  give  the  same  reaction  as 
psychoses  in  which  such  lesions  have  not  yet  been 
demonstrated.  Much  1  therefore  concludes  that  in 
both  cases  the  same  substance  circulates  in  the 
blood,  and  that,  moreover,  in  both  this  substance 
is  derived  from  a  degeneration  of  the  nerve  tissue. 

1  Much,  Die  Immunitatswissenschaft.      C.    Kabitzsch,  Wiirz- 
burg,  1911.         i 


210  APPENDIX 

Weil's  Cobra  Venom  Test  in  Syphilis 

In  studying  the  varying  resistance  of  red  blood 
corpuscles  to  haemolytic  agents,  Weil 1  noted  that 
the  corpuscles  of  syphilitics  were  regularly  more 
resistant  to  the  action  of  cobra  venom  than  those 
of  normal  individuals.  Just  what  is  the  cause  of 
this  increased  resistance  is  not  entirely  clear.  It 
is  known  that  syphilis  attacks  the  lipoids  of  the 
body,  and  that  the  amount  of  lecithin  which  can 
be  extracted  from  the  tissues  is  less  in  syphilitic 
conditions  than  in  normal  individuals.  The  in- 
creased resistance  has  therefore  been  thought  to 
be  due  to  a  decrease  in  the  lecithin  content  of  the 
red  blood  corpuscles. 

In  testing  the  corpuscles  the  blood  is  collected 
in  citrated  salt  solution,  and  then  washed  by 
repeated  centrifugalization  in  order  to  thoroughly 
remove  the  serum.  Even  slight  traces  of 
serum  interfere  decidedly  with  the  reaction.  The 
washed  cells  are  made  up  into  a  four  per  cent 
suspension  in  0.9  per  cent  common  salt  solution. 
Centrifugalization  is  done  in  very  accurately  grad- 
uated centrifuge  tubes,  as  the  accurate  dilution  of 
the  cells  is  a  matter  of  very  great  importance. 
The  cobra  venom,  which  is  kept  in  the  dried  con- 

1  Weil,  Richard,  Journal  of  Infectious  Diseases,  Vol.  vi,  Nov., 
1909.  Proceedings  Society  Exp.  Biology  and  Medicine,  Vol.  vi 
and  Vol.  vii. 


APPENDIX  211 

dition,  is  made  up  very  accurately  in  small  quan- 
tities into  a  0.05  per  cent  solution  in  0.9  per  cent 
solution  of  common  salt.  From  this  stock  solution, 
higher  dilutions  are  prepared  as  required,  and  are 
kept  at  a  constant  low  temperature.  In  carrying 
out  the  test  four  solutions  are  prepared,  namely, 
i  in  10,000,  20,000,  30,000,  and  40,000.  One  cc. 
of  the  corpuscle  suspension  is  added  to  one  cc.  of 
the  venom  solution.  The  mixtures  are  incubated 
for  one  hour  at  40°  C.,  after  which  a  preliminary 
inspection  will  almost  invariably  reveal  the  final 
results  of  the  tests.  At  this  stage,  cells  showing 
no  haemolysis  with  i  to  10,000  venom  are  strongly 
positive,  cells  showing  no  haemolysis  with  i  to 
20,000  are  positive,  cells  showing  very  slight  hsemol- 
ysis  at  i  to  30,000  are  weakly  positive;  cells  show- 
ing moderate  destruction  at  i  to  30,000  are  negative, 
and  cells  showing  the  least  appreciable  trace  of 
haemolysis  at  i  to  40,000  are  strongly  negative. 
After  the  preliminary  reading  the  mixtures  are 
again  shaken  and  then  kept  in  the  ice-box  over 
night,  the  final  readings  being  made  on  the  follow- 
ing morning.  The  figures  here  given  are  merely 
those  of  a  particular  specimen  of  cobra  venom; 
it  is  possible  that  other  samples  might  differ  in 
strength.  At  all  events  a  preliminary  titration 
against  corpuscles  derived  from  cases  of  known 
syphilis  and  controls  suffices  to  determine  the 
values  for  any  given  sample.  Inasmuch  as  the 


212  APPENDIX 

strength  of  the  dried  venom  does  not  vary,  this 
preliminary  titration  gives  a  constant  standard, 
which  is  practically  permanent,  since  one  gram  of 
venom  suffices  for  about  5000  complete  tests.  The 
standardization  is  different  for  infants  as  compared 
with  adults,  for  the  red  corpuscles  of  infants  and 
young  children  haemolyze  much  more  readily  than 
those  of  adults.  The  cells,  however,  acquire  the 
same  degree  of  resistance  as  do  those  of  adults  in 
disease. 

Weil  states  that  the  cobra  venom  reaction  appears 
to  have  certain  advantages  over  the  Wassermann 
reaction.  It  is  much  simpler  and  the  labor  much 
less.  Less  blood  is  required,  so  that  a  few  drops 
from  the  lobe  of  the  ear  suffice  for  a  reaction;  this 
is  of  some  importance  in  infants.  Cases  of  scarlet 
fever  and  of  leprosy  do  not  offer  a  source  of  con- 
fusion. The  reaction  is  more  marked  in  old, 
apparently  dormant  cases.  The  reaction  persists 
much  longer  after  mercurialization,  thus  offering  a 
further  diagnostic  and  therapeutic  test.  The  reac- 
tion is  possible  in  cases  of  jaundice,  whereas  the 
Wassermann  is  not. 

Antitrypsin  Determinations. 

We  have  already  pointed  out  that  the  animal 
body  responds  to  the  injection  of  ferments  by 
the  production  of  antiferments.  Considerable  in- 


APPENDIX  21- 

vy 

terest  has  been  aroused  by  the  discovery  that  in 
certain  diseases,  especially  cancer,  the  antitrypsin 
content  of  the  patient's  serum  is  markedly  in- 
creased. In  cancer  this  increase  is  noted  in  about 
90  per  cent  of  the  cases.  The  antiferment  action 
is  not  entirely  specific,  but  extends  to  other  pro- 
teolytic  ferments,  and  particularly  to  the  ferment 
of  leucocytes.  At  the  present  time,  therefore,  a 
marked  increase  in  the  antitryptic  power  of  a 
patient's  serum  is  taken  to  indicate  an  increased 
parenteral *  destruction  of  proteid  in  the  body.2 
The  original  method  of  demonstrating  the  presence 
of  this  antitrypsin  was  by  placing  drops  of  pro- 
teolytic  ferment  (trypsin)  on  the  surface  of  a  plate 
of  Loeffler's  serum,  and  causing  the  development  of 
small  concavities  owing  to  the  digestion  of  the 
medium.  The  addition  of  inhibiting  serum  to  the 
drops  was  able  to  prevent  the  formation  of  -  the 
concavities.  A  more  convenient  and  accurate 
method  is  the  one  developed  by  v.  Bergmann  and 
Meyer.  This  depends  on  the  digestion  of  a  per- 
fectly clear  solution  of  casein.  If  all  the  casein  has 
been  digested,  the  addition  of  acid  is  obviously 
unable  to  precipitate  any  casein  from  solution. 
On  the  other  hand,  if  the  acid  causes  clouding  or 
precipitation,  it  follows  that  all  the  casein  was  not 

1  Other  than  intestinal. 

2  See  the  excellent  digest  of  the  work  on  this  subject  in  Jahres- 
bericht  der  Immunitatsforschung,  Bd.  V,  1909,  Abteilung  I,  page 
58. 


APPENDIX 

digested.  It  is  evident  that  the  quantity  of  trypsin 
required  to  digest  a  given  amount  of  casein  can  be 
exactly  determined,  and  that  by  employing  grad- 
uated amounts  of  the  inhibiting  serum  accurate 
determinations  of  the  antitryptic  content  can  be 
made. 


INDEX  OF  AUTHORS 


Abel,  17,  100 
Anderson,  142,  160 
Altmann,  195 
Aronson,  5 
Arrhenius,  28 
Arthus,  141 
Ascoli,  208 
Atkinson,  18 
Auer,  150 

Bail,  93,  158 

Banzhaf,  19,  151,  153 

Beebe,  125 

von  Behring,  i,  5,  170 

Belfanti,  18 

von  Bergmann,  013 

Besredka,  82,  84 

Blumenthal,  198 

von  Bokay,  144 

Bolduan,  135 

Bordet,  28,  40,  52,  56,  67,  87 

96,  108,  185,  207 
Braun,  194 
Bruck,  72,  196 
Buchner,  i,  6,  50,  75,  78,  93 
Buxton,  104 

Calmette,  18,  137 
Carbone,  18 
Castellani,  46 


Citron,  158,  198 
Crile,  205 

Dallera,  143 

Delezenne,  121 

Deutsch,  1 66 

Dieudonne,  18,  113 

Doerr,  149,  158 

Donath,  94 

Douglas,  128 

von  Dungern,    16,   54,  79,  85, 

Qi,  I25 
Durham,  37 

Ehrlich,  6,  u,  13,  16,  23,  39, 
47,  58,  67,  84,  87,  92,  97, 
112,  159,  166,  179 

Eisenberg,  39,  in 

Epstein,  205 

Famulener,  153 
Field,  43 
Fischer,  9 

Fleischmann,  195,  197 
Flexner,  137 
Floyd,  178 
Fodor,  50 
Ford,  17 
Fornet,  196 
Friedberger,  42,  156 


216 


INDEX  OF  A  UTHORS 


Gay,  73,  88,  96,  104,  146,  196, 

207 
Gengon,  40,  71,  99,   163,   185, 

196 

Gibson,  18 
Grassberger,  29 
Gruber,  36,  93 
Griinbaum,  37 
Gscheidlen,  50 

Haeckel,  127 
Hahn,  93 
Hamburger,  148 
Hektoen,  130 
Hericourt,  141 
Heubner,  144 
Hiss,  177 
Holzmann,  208 

Jackson,  126 
Jansky,  35 
Jenner,  169 
Jordan,  150 

Kitasato,  i 
Kleine,  166 
Knorr,  2,  5 
Kraus,  108 
Kyes,  137 

Landois,  74,  143 
Landsteiner,  35,   54,   94,    123, 

194,  196 
Leblanc,  in 
Leclainche,  109 
Ledingham,  20 
Leishman,  131 
Levaditi,  194,  198 
Lewis,  150 
Loffler,  100 
Lucas,  178 


Manwaring,  139 

Marie,  194,  198 

Marrassini,  125 

Martin,  5 

Marx,  86 

Meier,  194 

Mertens,  109 

Mesnil,  166 

Metchnikoff,    38,    52,    58,    63, 

82,  84,  91,  93,  99,  121,   i27; 

1 60 

Meyer,  213 
Michaelis,  196 
Moreschi,  97 

Morgenroth,    18,    29,   71,    109 
Moro,  148 
Moss,  35,  98 
Moxter,  86,  93,  123 
Much,  208 
Muir,  67 

Muller,  63,  91,  194 
Myers,  109 

Neisser,  72,  100,  196 

Neudorfer,  143 

Neufeld,  129 

Nissen,  74 

Noguchi,  137,  195,  199,  200 

Nolf,  91 

Nuttall,  50,  74,  no,  115 

Obermayer,  in 
Ottenburg,  205 
Otto,  141 
Opie,  162 

Park,  2,  74 
Pasteur,  169,  171 
Pearce,  126 
Pfaundler,  38 


INDEX  OF  A  UTHORS 


217 


Pfeiffer,  37,  51,  86,  93 

Pick,  19,  109,  in 

von  Pirquet,  142,  147,  164 

Plant,  192 

Porges,  194 

Potzl,  194 

Richet,  141 
Rimpau,  129 
Rosenau,  142 
Roux,  5 
Russ,  149 

Sachs,  73,  137,  195 
Salge,  161 
Salmon,  171 
Schattenfroh,  29,  93 
Schick,  142 
Schultz,  150 
Schiitze,  109,  113,  120 
Smith,  141,  161,  171 
Southard,  146 
Spiro,  109 
Steinhardt,  151 
Stern,  109,  113,  197 
Streng,  207 


Tchistowitch,  107 
Touissant,  171 
Traube,  50 

Uhlenhuth,  109,  113 

Vallee,  109 
Volk.  39 

von  Wassermann,  13, 1 8,  72,  7- 
86,  92,  93,  99,  109,  113,  15; 
185,  192 

Wechsberg,  100 

Wechselmann,  183 

Weichardt,  208 

Weigert,  10 

Weil,  194 

Weil,  R.,  210 

Welch,  165 

Wernicke,  5 

Widal,  37 

Wile,  198 

Wright,  128,  131,  173 

Zinsser,  178 
Ziilzer,  109 


SUBJECT    INDEX 


PA.GE 

Abrin 21 

Absorption,  in  study  of  agglutinins 46 

of  complement,  see  Bordet-Gengou 71 

and  Neisser-Wechsberg 100 

Active  immunization,  methods 171 

Adsorption,  as  applied  to  toxin-antitoxin 30 

elective  character 31 

Agglutination,  in  typhoid  fever 37 

influence  of  dilution  on 34 

phenomenon  of 32 

purpose  of 36 

specificity  of 44 

Agglutinin 32 

against  trypanosomes 34 

effect  of  heat  on 39 

nature  of 39 

Agglutinoid 39 

Aleuronat,  in  production  of  Icucocytic  exudates 177 

Alexin,  see  also  complement 56 

Allergy 147 

Amboceptor,  definition 63 

unit 20 1 

Antibacterial  sera,  practical  value  of 104 

Antibodies,  cells  concerned  in  production  cf n 

Anticomplement 86,  88 

views  concerning '. .  . .  96 

Anticomplementary  serum,  specificity  of 90 

Anticy  totoxin 122 

Antigen,  definition  of 16 

for  syphilis  test 190 

Antihaemolysins,  their  nature 86 

219 


220  SUBJECT  INDEX 

PAGE 

Antiprecipitin 119 

Antiserum,  for  snake  venoms 137 

Antitoxic  globulins 1 8 

Antitoxin,  action  on  toxins 18 

diphtheria,  production  of 2 

diphtheria,  testing  of 5 

historical i 

nature  of r.  17 

quantitative  relation  to  toxin 31 

relation  to  toxin 22 

unit 22 

Antitrypsin,  determination  of 212 

Antivenins 139 

Anaphylactic  shock 142 

pathology  of 150 

prevention  by  chloral  hydrate 153 

Anaphylatoxin 157 

Aggressins 158 

Anaphylaxis,  historical 141 

relation  to  precipitins 148 

relation  to  serum  therapy , 151 

theories  of 144 

Anthrax,  symptomatic,  toxin  of 29 

Antianaphylaxis 1.^3 

Asthma,  relation  to  anaphylaxis 151 

Atrepsy 166 

Autolysins 97 

Bacterial  precipitins 108 

Bactericidal  serum,  clinically 105 

Bacteriolysins,  historical 50 

Bacteriolysis,  of  cholera  spirilla 51 

Bacteriotropic  substances 127 

Blood,  biological  reaction  for 73,  1 13 

examination  for  transfusion 204 

its  germicidal  power 50,  74 

cells,  agglutination  of 34 

relationship,  by  means  of  precipitin  test no 

test,  Neisser-Sachs 73 


SUBJECT  INDEX  22i 


Bordet-Gengou  phenomenon 71 

Brain  tissue,  effect  on  tetanus  toxin 13 

Cancer,  immunization  against 125 

Casein,  precipitin  against 108 

Chemoreceptors 181 

Chloral  hydrate,  to  prevent  anaphylactic  shock 153 

Cholera  spirilla,  Pfeiffer's  test 51 

Clumping,  see  Agglutination .  32 

Cobra-lecithid 139 

Cobra  venom 137 

as  test  in  syphilis 210 

in  haemolysis 208 

Colloids,  coagulability  of 42 

in  the  study  of  agglutination 42 

Common  receptors 83 

Complement 64 

absorption  by  precipitates 96 

absorption  of,  see  Bordet-Gengou 71 

artificial  increase 92 

definition  of . 63 

deflection  of i  oo 

effect  of  heat  on 94 

effect  of  phosphorus  poisoning  on 91 

multiplicity  of 70 

source  of 93 

structure  of 94 

Complementoid 94 

Complementophile  group,  Bordet's  views  concerning 67 

Conglutination 207 

Copula,  definition  of 63 

Crotin 21 

Cytotoxins,  definition 121 

produced  by  nucleo-proteid  injections 125 

specificity  of 124 

Desmon,  definition  of 63 

Deviation  of  complement 100 

Dissociation,  in  toxin-antitoxin  action 29 


222  SUBJECT  INDEX 


Dosage,  of  lytic  sera ..." 100 

Dyeing,  analogous  to  antitoxin  reaction. 30 

Electric  charge,  of  agglutinins 43 

Electrolytes,  in  relation  to  agglutination 41 

Endotoxins,  relation  to  infection 158 

Endotoxin  theory,  applied  to  anaphylaxis 145 

Epithelium,  cytotoxin  for 124 

serum  against 85 

Fractional  neutralization,  in  study  of  antitoxins 30 

Ferments,   in  leucocytes 162 

Globulin,  antitoxic 18 

Group  agglutination 44 

Gruber- Widal  reaction 37 

Haemagglutinins 34 

Haemolysin,  discovery  of . . 52 

Haemorrhagin,  of  snake  venom 138 

Haptins,  definition  of 16 

Haptophore  group,  nature  of 7 

Heat,  effect  on  serum 40,  96 

Hypersensitization,  to  serum 142 

Immediate  reaction 145 

Immune  bodies,  site  of  production 86 

Immune  body ,  .  .  .  64 

nature  of 82 

Immunity,  acquired 160 

hereditary  transmission 160 

mechanism  of 162 

natural,  variations  in 159 

relation  of  anaphylaxis  to 163 

varieties  of 158 

Immunization,  active  contrasted  with  passive 161 

active,  methods  of 171 

against  diphtheria  toxin.  .  .- 3 

with  partially  neutralized  toxins 12 


SUBJECT  INDEX  223 

PAGE 

Inactivated  serum .  .  .  .  : 54 

Index,  opsonic 132 

Infection,  nature  of 154 

relation  of  anaphylaxis  to 156 

relation  of  virus  to 154 

Inflammation,  Opie's  researches  on 162 

Interbody  of  normal  serum 77 

Isoagglutinin,  occurrence  of  in  normal  human  sera 35 

relation  to  homologous  transfusion 36 

Isolysins,  occurrence  of  in  man 98 

nature  of 97 

Isoprecipitin 120 

Lactoserum. .  .  i 108 

Lecithid,  of  cobra  venom 139 

Leucocytes,  as  source  of  complement 93 

ferments  in 162 

phagocytic  action  of 127 

Leucocyte  extract,  in  treatment  of  infections 177 

Leucotoxin 121 

Liver,  role  in  production  of  complement 91 

L0  and  L| 23 

Lysins,  relation  to  agglutinins 36 

Mastic,  agglutination  of 41 

Meiostagmin  reaction 208 

Milk,  human  and  bovine  tested  biologically 112 

Much-Holzmann  reaction 208 

Mussels,  toxin  of 141 

Negative  phase 133 

Neisser-Sachs  blood  test 73 

Neisser-Wechsberg  phenomenon 100 

Nephrotoxin 126 

Neurotoxin 122 

of  snake  venom 138 

Noguchi's  test  for  syphilis 200 

Normal  serum,  agglutinating  poweY  of 34 

contrasted  with  specific  immune  serum. 79 


224  SUBJECT  INDEX 


PAGE 


Normal  serum,  its  haemolytic  and  bacteriolytic  action.       .  ;  73 

Nucleo-proteid,  for  immunization 125 

Opsonic  index,  clinical  value  cf 135 

nature  of 131 

Opsonin,  affinity  of,  for  bacteria 129 

histoi  ical 127 

structure  of 130 

Organotropism 179 

Osmotic  pressure,  of  serum,  in  infections 208 

Parasitotropism 179 

Parenteral  digestion,  of  proteid 164 

Partial  immune  bodies 82 

Partial  saturation  method 24 

Pfaundler's  thread  reaction 38 

Pfeiffer's  phenomenon 51 

Phagocytosis 127 

Phosphorus  poisoning,  effect  on  complement  production.  . .  91 

Physical  chemistry,  applied  to  toxin-antitoxin  reaction.  ...  28 

Precipitates,  relation  to  anti-complement  action 96 

Precipitins,  definition  of , 107 

in  relation  to  animal  species no 

method  of  immunization 114 

nature  of in 

specificity  of 109 

Precipitin  test,  for  blood 116 

various  applications  of 1 18 

Preparator,  definition  of 63 

Pro  zone,  in  agglutination 39 

relation  of  colloids  to , 43 

Proteid,  cleavage  of 157 

parenteral  digestion  of 164 

Protoxoid 26 

Psychoses,  serum  diagnosis  in 209 

Ptomaines 20 

Rash,  from  serum  injections 143 

Receptors,  common 83 


SUBJECT  INDEX  22'5 


Receptors,  nature  of g 

various  orders  of 47 

Reversible  reactions  in  toxin-antitoxin  combination 29 

Ricin 21 

Calts,  their  relation  to  agglutination 41 

Salvarsan,  chemistry  of 182 

principles  governing  treatment  by 1 79 

Serum,  active  and  inactive 54 

against  epithelium 125 

antibody  content  of  compared  to  plasma 163 

effect  of  heat  on 40 

mode  of  collection 4,  115 

Serum  diagnosis,  in  typhoid  fever 37 

Serum-fast  trypanosomes 166 

Serum  rashes 143 

Sessile  receptors 14 

Side-chain  theory,  applied  to  antitoxins.  .  . ;. 6 

applied  to  bacteriolysins  and  haemolysins 68 

Snake  venom 137 

Specific  therapy,  principles  of 179 

Specificity,  cause  of 65 

nature  of 65,  124 

of  agglutination 45 

Spectrum,  Ehrlich's  so-called. 25 

Spermatoxin 123 

Split  products,  in  anaphylaxis 146 

Stimulins 128 

Substance  fixatrice 63 

sensibilatrice 55 

Syntoxoid 27 

Syphilis,  cobra  venom  test  in 210 

serum  diagonsis  of 185 

specific  treatment  of .* 179 

tests  for 196 

Tetanus  toxin,  affinity  for  brain  tissue 13 

Thread  reaction 38 

Toxin -antitoxin  reaction 22-31 


226  SUBJECT  INDEX 

PAGE 

Toxin,  absorption  of  antitoxin  by 31 

diphtheria,  preparation  of 2 

relation  to  antitoxin 22 

Toxins,  characteristics  of 20 

Toxoids,  nature  of 6 

various  kinds  of 24 

Toxons 24 

Toxophore  group,  its  influence  in  immunization 1 6 

nature  of 7 

Transfusion,  blood  tests  in 204 

relation  of  isoagglutinins  to 36 

Trypanosomes,  agglutinins  against 34 

serum-fast 1 66 

Typhoid  fever,  Gruber-Widal  reaction  in 37 

Uhlenhuth  blood  test 1 16 

Vaccines,  bacterial 169 

bacterial,  doses 175 

Vaccine  therapy 173 

Venoms,  of  snakes 137 

Viscosity  of  blood,  relation  to  serum  reactions 40 

Wassermann  test,  for  syphilis 185 

Weigert's  overproduction  theory 9 

Welch's  hypothesis 165 

Widal  reaction 37 

Zootoxins 21 

Zymotoxic  group 94 


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